Methods and compositions for controlling insects

ABSTRACT

Compositions and methods for controlling insects by co-expressing an amino acid oxidase and a second enzyme that provides insecticidal activity when present in a mixture with the amino acid oxidase are disclosed. Also disclosed are DNA and protein sequences, and transformed microorganisms and plants useful for achieving such insect control.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application Ser.No. 60/044,504, filed Apr. 21, 1997.

FIELD OF THE INVENTION

This invention relates to compositions and methods for controllingcoleopteran insects by use of two proteins in combination which may beapplied directly to the plant or produced thereon by microorganisms orby genetically modifying the plant to produce the proteins, to genesencoding these proteins, to methods for identifying such genes andproteins, and to recombinant microorganisms and plants capable ofexpressing these genes for use in controlling plant infestation by thetarget coleopteran insects.

BACKGROUND OF THE INVENTION

The control of insect pests by naturally occurring proteins is a wellestablished practice. The most commonly used insect control proteins arethe endotoxins derived from Bacillus thuringiensis (B.t.) that are usedto control both lepidopteran and coleopteran insect pests. Expression ofthese proteins in transgenic plants also confers protection againstcertain insect pests (Barton et al., 1987; Fischhoff et al., 1987 Perlaket al., 1990; Vaeck et al., 1987).

A variety of insect pests that cause significant economic losses werenot previously known to be controlled by B.t. endotoxins. Boll weevil(BWV), Anthonomus grandis, corn rootworm (CRW), Diabrotica spp., andwireworm (WW), Melanotus spp. are examples of coleopteran insect peststhat inflict significant crop damage yet, until recently were not knownto be controlled by known B.t. endotoxins. Thus, it would be useful toidentify new insecticidal proteins which, alone or in combination, areable to control these coleopteran insects. Furthermore, it would beuseful to identify new insecticidal proteins with different modes ofaction to delay the development of B.t. endotoxin resistance incoleopteran pests such as the Colorado potato beetle (CPB), Leptinotarsadecemlineata (Say), that are currently controlled by certain B.t.endotoxins (Krieg et al., 1983).

Preparations of enzymes from several different sources are availablefrom Sigma Chemical Company (St. Louis, Mo.) and other suppliers. Aminoacid oxidases can also be obtained from sources including, but notlimited to snake venom, mammalian, and avian sources (Bright and Porter,1975). Lysine and other amino acid oxidases (E.C. 1.4.3.2) are naturallyproduced by micro-organisms such as Trichoderma sp., Neurospora sp.,Penicillium sp., and Proteus sp. (Kusakabe et al., 1979; 1980; Niedermanand Lerch, 1990; Knight, 1948; Stumpf and Green, 1944). Although lysineoxidase has been shown to have antitumor activity (Kusakabe et al.,1979; Id., 1980), there have been no reports of insecticidal activityassociated with this enzyme. Also, there have been no reports ofinsecticidal activity being associated with an amino acid oxidase enzymewhen combined with any other compound. However, we have unexpectedlyfound that a composition comprising a lysine oxidase and a previouslyunidentified M_(r) 50,000 protein yield potent insecticidal activitywhen combined in a mixture and ingested by an insect. The M_(r) 50,000protein is described herein as a tedanalactam synthase shown herein tohave at least one enzyme activity in which Δ1-piperideine-2-carboxylateis converted to tedanalactam. Described herein are methods for using acombination of lysine oxidase and tedanalactam synthase to controlinfestation of plants by insect pests.

SUMMARY OF THE INVENTION

This invention relates generally to novel compositions and methods forthe control of undesired insects. It is therefore a particular object ofthe present invention to present materials and methods used in thepreparation of compositions and plants capable of controlling insectinfestation when ingested by the insect. It is also an object of thepresent invention to provide protein compositions capable of controllingBWV, CRW, WW, CPB or other insect pests, and genes useful in producingsuch proteins. It is a further object of the present invention toprovide genetic constructs for and methods of inserting such geneticmaterial into microorganisms and plant cells. It is another object ofthe present invention to provide transformed microorganisms and plantscontaining such genetic material. Still another object of this inventionrelates to methods and reagents such as polynucleotides and antibodies,and the use of such methods and reagents in kit form, for detecting theindividual molecules which comprise the active compositions as notedherein. In addition, variants of the molecules which comprise the activecompositions are also contemplated by this invention.

Among the several advantages found to be achieved by the presentinvention, therefore, may be noted the provision of a compositioncontaining at least two proteins, lysine oxidase enzyme and tedanalactamsynthase enzyme, which is capable of controlling insects, particularlycoleopteran insects. These two proteins cause mortality and stunting oflarvae of coleopteran insects when co-ingested. The proteins may beapplied directly to plants or introduced in other ways such as throughthe application of plant-colonizing microorganisms or by transformedplants generated using recombinant DNA methods wherein the recombinantplants express genes encoding these enzymes.

In accomplishing the foregoing, there is provided, in accordance withone aspect of the present invention, a method of controlling insectinfestation of plants comprising providing a composition containing atleast a lysine or amino acid oxidase along with a second enzyme, whichis preferably a tedanalactam synthase, for ingestion by the insect. Itis apparent that neither protein alone is able to confer anyinsecticidal activity. However, it is the combination of the amino acidoxidase along with the second enzyme which is effective in conferinginsecticidal activity upon ingestion of such a composition by an insect.The composition, upon ingestion by the insect, contains a sufficientinsecticidal amount of the proteins, such that the insect is unable tosurvive or is rendered incapable of causing further damage to a plant towhich the composition has been applied. The composition contains a firstenzyme which is a lysine oxidase enzyme, and a second enzyme which iscapable of converting Δ1-piperideine-2-carboxylate to tedanalactam. Theproteins in the composition are preferably isolated from extracts offungal species fermentations in which the extracts have been shown toexhibit insecticidal activity. The fungal species herein which producesan insecticidally effective extract composition was determined to be aTrichoderma species of fungi, and in particular a Trichoderma viride.Another Monsanto Company fungal isolate was designated as Trichodermasp. F22844 also produces an insecticidally effective extractcomposition. The genes encoding the proteins in the illustrativecomposition are therefore preferably isolated from a Trichoderma speciesof fungi, however, other uncharacterized fungal species are believed tocontain at least a lysine oxidase gene and a second protein which, incombination provide efficacious insecticidal activity.

The composition can contain as the second enzyme a protein which isapproximately 50,000 Da, which is also recognized by one skilled in theart as a protein or enzyme which is approximately M_(r) 50,000. It isbelieved that the second enzyme can be isolated from any number ofspecies, however it is preferably isolated from a species which producescompounds which exhibit coleopteran insecticidal activity, and morepreferably isolated from a fungal species. It is also believed that anyfungal species which exhibits coleopteran insecticidal activity and alsoproduces a lysine oxidase may also produce a second enzyme which incombination with the lysine oxidase confers effective insecticidalactivity when ingested by target insect. Furthermore, any species whichproduces a lysine oxidase and which also contains a gene whichhybridizes under stringent conditions to a Trichoderma species geneencoding an approximately M_(r) 50,000 Da protein which convertsΔ1-piperideine-2-carboxylate to tedanalactam may confer effectiveinsecticidal activity when a composition containing both enzymes isingested by a target insect. The property of convertingΔ1-piperideine-2-carboxylate to tedanalactam may be independent of theproperty which, in combination with an amino acid oxidase confersinsecticidal activity upon the composition when ingested by the insect.It is intended that the composition not be limited to a combination ofan amino acid oxidase and a tedanalactam synthase, but conceivably couldalso include the combination of a gene encoding an amino acid oxidaseand a gene encoding a tedanalactam synthase, together with all necessarygenetic regulatory elements required for expression, includingrepression and activation, transcription and translation, andpost-transcriptional and post-translational modification signalsincorporated therein. The genes as described could also be presenteither alone or in combination with each other on a single replicon.

The composition which confers coleopteran insecticidal activity isdirected preferably to coleopteran species selected from the groupconsisting of Diabrotica species, Melanotus species, Leptinotarsaspecies, and Anthonomus species. Moreover, the composition is directedto controlling insects selected from the group consisting of boll weevil(BWV), corn rootworm (CRW), corn wireworm (WW), and the Colorado potatobeetle (CPB).

The compositions in particular can contain the indicated enzymes in amixture in which the molar ratios of the two enzymes are generally suchthat effective insect control is manifested. Insect control can beeffected when the amino acid oxidase and the tedanalactam synthase arepresent within the composition in molar ratios of about 100:1 to about1:1 respectively, or when the ratios are about 10:1 to about 1:1,respectively, or when the ratios of the two proteins are present fromabout 1:10 to about 1:1, or when the ratios of the two proteins arepresent from about 1:100 to about 1:1, respectively. In addition,effective concentrations of these proteins in a composition in which theproteins are each present from about one part per million to about 10parts per million are effective in conferring insecticidal activity andcontrol. The most effective insecticidal is activity is conferred whenthe proteins are each present in a composition from about one part permillion to about 20 parts per million.

Another aspect of the present invention provides the structural geneswhich encode the enzymes which are the active components in theinsecticidal compositions. Briefly, the genes can be isolated fromgenomic DNA and from cDNA molecules which are obtained by isolating MRNAfrom species which are shown to produce these enzymes. The structuralgenes encoding these enzymes, which may also be isolated as proenzymesor precursor proteins, preferably are identified by first isolating theactive components or enzymes from extracts of organisms which producethese enzymes. Isolated enzymes can be digested with proteolyticenzymes, and amino acid sequences of proteolytic peptide fragments canbe characterized. Redundant nucleotide probes corresponding to thecharacterized peptide fragments can be produced based on the deducedamino acid sequences, and used as probes or primers for identifying oramplifying particular segments of mRNA, cDNA, or genomicpolynucleotides. Full length MRNA, full length CDNA, and uninterruptedfull length genes can be further identified and isolated.

In accordance with other aspects of the present invention, there areprovided methods and compositions for producing genetically transformedplants which express an amount of a lysine or other amino acid oxidasealong with a second enzyme or tedanalactam synthase effective to controlcoleopteran insects. Recombinant plasmids have been produced whichcontain regulatory elements which function in plants for producingmessenger RNA molecules, from which the proteins of the presentinvention are translated. Expression cassettes are disclosed whichcontain various elements alone or in combination for enabling theproduction of the amino acid oxidase or the tedanalactam synthase.Specifically, the amino acid oxidase gene is provided in a cassettecomprising a polynucleotide sequence flanked 5′ by a promoter whichfunctions in plants to cause the production of an RNA sequence isoperably linked to an intron and a DNA sequence which functions inplants as a targeting signal or transit peptide and flanked 3′ by a DNAsequence which functions in plants to cause the addition of a 3′non-translated polyadenylated nucleotide sequence to the 3′ end of theRNA is fused 3′ to the amino acid oxidase gene so that the expression ofthe cassette is under the control of the promoter. There are numerousalternatives to this construction, some of which are provided herein inspecific examples. For example, the intron and targeting sequence can bereplaced by a 5′ non-translated leader sequence; or the non-translatedleader can be removed; the intron can be inserted between thenon-translated leader and the oxidase gene or between the leader and thetargeting sequence. The tedanalactam synthase can be assembled in asimilar fashion, and specific examples are provided herein. Anexpression cassette for producing an amino acid oxidase can be combinedinto a single vector along with a cassette for producing a tedanalactamsynthase so that delivery of both cassettes for simultaneous expressioneither in a plant or other organism such as a bacterium or fungi is alsocontemplated. Also, in a plant it is possible to express one of thecassettes in one tissue type, for example in roots, and express theother cassette in another tissue type, for example in leaves. It mayalso be possible to produce the proteins separately temporally orspatially, but in the same tissue type. For example, expression of onecassette in young leaves and the other cassette later in the same leavesis contemplated, however co-expression is normally desirable. Theexpression cassette can be designed to function in plants by using plantspecific regulatory elements such as promoters, introns, targetingsequences, non-translated leaders, and 3′ polyadenylation sequences. Theexpression cassette can also be designed to function in prokaryoticsystems as contemplated and described herein, also by using prokaryoticspecific regulatory elements. The cassettes described herein can beinserted into plants by high velocity DNA coated particle projectilebombardment, by naked DNA protoplast transformation, or by bacterialmediated methods known in the art.

In describing this particular embodiment of the invention, it should beunderstood that expression of the amino acid oxidase, which can also bea precursor or proenzyme, and tedanalactam synthase can be controlled bytwo independent promoters from two separate and independenttranscriptional units. It should also be understood that a singlepromoter could be used to drive expression of a single transcriptionunit containing an in frame translational fusion of both proteins. Thehybrid polyprotein could then be post-translationally cleaved to yieldboth proteins by previously described schemes (Halpin and Ryan, WO95/17514). Another advantage achieved by the present invention providesa peptide fusion to be produced from the genes encoding the two enzymeswherein the coding sequences of the two genes are fused in frame toallow for the expression of a recombinant gene encoding an in-frametranslational peptide fusion of the amino acid oxidase and thetedanalactam synthase. The fusion can be one in which either enzyme isamino terminal with respect to the other. The fusion can bepost-translationally cleaved by a plant endogenous endoprotease toproduce an insecticidally active composition in the plant tissues sothat lysine oxidase and tedanalactam synthase are present as separateand individual molecules. Alternatively, the fusion can bepost-translationally cleaved by an endogenous insect endoprotease,generally found within the midgut of contemplated insect targets, sothat the cleavage of the fusion protein produces an insecticidallyactive composition while within the midgut of the feeding insect.

In keeping with this aspect of the present invention, is the provisionfor a variety of promoters for transcriptional initiation and expressionof the contemplated genes, in particular in plants. A number ofpromoters which are active in plant cells have been described in theliterature. Such promoters may be obtained from plants or plant virusesand include, but are not limited to the nopaline synthase (NOS) andoctopine synthase (OCS) promoters, which are carried on tumor-inducingplasmids generally found within virulent and non-virulent strains ofAgrobacterium tumefaciens, the cauliflower mosaic virus (CaMV) 19S and35S promoters, the light-inducible ribulose 1,5-bisphosphate carboxylasesmall subunit promoter(ssRUBISCO), and the Figwort Mosaic Virus 35Spromoter (FMV). All of these promoters have been used to create varioustypes of DNA constructs which have been expressed in plants (see forexample Barry et al. U.S. Pat. No. 5,463,175, which is hereinincorporated by reference). Particularly desirable promoters which arecontemplated because of their constitutive nature are the CauliflowerMosaic Virus 35S (CaMV35S) and the Figwort Mosaic Virus 35S (FMV35S)promoters which have previously been shown to produce high levels ofexpression in most plant organs. Other preferred promoters are rootenhanced or root tissue specific promoters such as the CauliflowerMosaic Virus derived AS4 promoter (also designated as the 4x as-1 or the4as-1 promoter), the tobacco RB7 promoter, or the rice RC2 promoter (Lamet al., 1991; Yamamoto et al., 1991; Xu et al., 1995). The root enhancedor root tissue specific promoters would be particularly preferred forthe control of corn rootworm (Diabrotica spp.) in transgenic cornplants. Other promoters are also contemplated which would direct tissuespecific targeted expression are also contemplated, for example intissue such as leaves, meristem, flower, fruit and organs ofreproductive character. IN addition, chimeric promoters are alsoenvisioned.

Other expression regulatory elements are considered to be of importance,especially in contemplation of transformed plant tissue or transformedplant cell expression. These elements comprise at least non-translatedsequences and introns. For example, transcriptional events leading toRNA production from the contemplated DNA constructs as set forth hereincould contain 5′ non-translated leader sequences. These sequences can bederived from new or existing promoters which are selected for geneexpression, and can be specifically modified so as to increasetranslational efficiency of the mRNA. A plant gene leader sequence whichhas been shown to be particularly useful in the present invention is thepetunia heat shock protein 70 leader (hsp70)(Winter et al., 1988). Ithas also been shown that introns are preferred for optimum expression inmonocotyledenous plants. Any number of introns could function for thispurpose, and without intending any limitation these could be selectedfrom the group consisting of the maize hsp70 intron and the rice actinintron. Another nontranslated regulatory element of particularimportance in plant systems are DNA sequences which function in plantsto cause the addition of a 3′ non-translated polyadenylated nucleotidesequence to the 3′ end of an RNA sequence produced as a result oftranscription from an indicated promoter sequence. These particularnon-translated sequences are also commonly known as polyadenylationsequences or signals.

A further embodiment provides for the targeted delivery of a geneproduct to a particular organellar compartment, such as a vacuole, amitochondrion, a chloroplast, a plastid, an endoplasmic reticulumcompartment, a Golgi compartment, or even the nuclear or nucleolardomains. Particular peptide sequences have been shown to be necessary inobtaining efficient delivery of protein products to these sites, inparticular signal peptides, signal sequences, and targeting sequences.Targeting signals or transit peptides contemplated herein, withoutintending to be limited to these, can be selected from the groupconsisting of a rice malate dehydrogenase amino terminal peroxisomaltargeting signal and a maize ATP synthase beta subunit mitochondrialtransit peptide.

A further embodiment of the present invention provides for the insertionof one or more of the contemplated expression cassettes along with anyexpression regulatory elements into the genome of a plant cell to form astable recombinant plant cell. In one embodiment the DNA inserted intothe plant genome would be comprised of a single cassette encoding afusion peptide formed from the gene fusions described above, along withregulatory elements necessary for expression of the gene fusion. Inanother embodiment, the DNA inserted into the genome would be comprisedof a single cassette encoding only an amino acid oxidase or atedanalactam synthase, along with any regulatory elements necessary forexpression of the gene. In the preferred embodiment, the DNA insertedinto the plant genome would be comprised of a first gene cassetteencoding an amino acid oxidase in which expression was controlled by afirst promoter, and a second gene cassette encoding a tedanalactamsynthase in which expression was controlled by a second promoter, alongwith any other necessary regulatory elements for expression of eithergene. This embodiment contemplates that the first and the secondpromoters can be identical in sequence and function, or they can bedifferent from each other, so long as each gene is expressed in anamount which provides a composition for controlling insect infestationof plants comprising a mixture of the enzymes when the mixture isingested by a susceptible insect.

A further embodiment of the present invention provides methods forgenerating plants which express an insecticidally effective amount of alysine or amino acid oxidase or proenzyme along with a tedanalactamsynthase. The methods utilize contemplated DAN expression cassettesdesigned for producing either or both enzymes separately or incombination inserted into plasmids. The plasmid DNAs can be directlyinserted into the genome of a plant by mechanical approaches, such asbiolistic methods or by protoplast fusion techniques. A preferred methodutilizes Agrobacterium mediated double border plant transformation,preferably using a DNA vector containing the desired expression cassetteor cassettes flanked by Ti plasmid border recombination sequences inorder to introduce the desired genes into the plant genome. Thetransformation procedure generally produces events which providestransformed plant cells selected on solid or in liquid media using anynumber of selectable markers known in the art, preferably glyphosateselectable markers such as GOX or EPSPS, antibiotic selectable markers,or others. Transformed cells obtained using these methods can be furtherregenerated to produce stably transformed genetically engineered plantswhich express insecticidally effective amounts of the amino acid oxidaseor the tedanalactam synthase.

There is also provided, in accordance with another aspect of the presentinvention, transformed bacterial and plant cells that contain DNAcomprised of the expressible gene cassettes as described above, alongwith appropriate control sequences necessary to provide for desired andappropriate expression of the coding sequences, producing insecticidallyeffective amounts of the enzymes. The control sequences can be any knownin the art to function in a particular cell or organelle type. It iscontemplated that the genes herein can be expressed in bacterialsystems, plant nucleolar compartments, plant nuclear compartments, andin plant mitochondrial and chloroplast compartments.

While particular examples of using the invention described herein tocontrol corn rootworm in corn or Colorado potato beetle in potato areprovided, it is understood that the methods of this invention could beapplied to provide insect protection, and more preferably coleopteraninsect protection, to plant species from the genera Fabaceae, Medicago,Trifolium, Vigna, Daucus, Arabidopsis, Brassica, Raphanus, Sinapis,Lycopersicon, Capsicum, Solanum, Nicotiana, Helianthus, Bromus,Asparagus, Panicum, Pennisetum, Cucumis, Lolium, Glycine, Triticum,Gossypium and Zea. In addition, forestry crop species from the generaPinus, Populus, Eucalyptus, Acacia, Silex, and Larix are also prone toimportant coleopteran pest infestation which may be controlled by themethods and compositions described herein. Also, turf grass species suchas St. Augustine (Stenotaphrum secundatum), Kentucky blue grass (Poapratensis), and creeping bentgrass (Agrostis stolonifera) among othersare susceptible to coleopteran pests such as white grub and the likewhich may also be controlled by the present invention. Insect pestswhich infest Roses (Rosa) and perennials such as Begonia, Pelagonium,Imaptiens, Tagetes, Viola, Petunia and Catharanthus and the like mayalso be subjects of the present invention.

FIGURE LEGENDS

FIG. 1 represents a plasmid map of pMON25061 which is a planttransformation vector containing a neomycin phosphotransferaseselectable marker under the control of a cauliflower mosaic virus 35Spromoter; and a tedanalactam synthase gene and lysine oxidase gene eachunder the control of separate root enhanced 4AS1 promoters.

FIG. 2 represents a plasmid map of pMON25049 which is an Agrobacteriummediated double border plant transformation vector containing a neomycinphosphotransferase selectable marker under the control of a cauliflowermosaic virus 35S promoter; a lysine oxidase gene fused to a petuniahsp70 leader sequence under the control of a figwort mosaic viruspromoter; and a tedanalactam synthase gene fused to a petunia hsp70leader sequence under the control of a figwort mosaic virus promoter.

FIG. 3 represents a plasmid map of pMON23671 which contains an 800 basepair CDNA fragment representing a portion of a lysine oxidase gene.

FIG. 4 represents a plasmid map of pMON23683 which contains a 650 basepair cDNA fragment representing a portion of the 3′ end of a lysineoxidase gene.

FIG. 5 represents a plasmid map of pMON23681which contains a 300 basepair cDNA fragment representing a portion of the 5′ end of a lysineoxidase gene.

FIG. 6 represents a plasmid map of pMON23680 which contains a genomicDNA fragment encoding a lysine oxidase gene.

FIG. 7 represents a plasmid map of pMON23684 which contains a genomicDNA fragment encoding a lysine oxidase gene.

FIG. 8 represents a plasmid map of pMON25421 which contains a cDNAfragment representing a partial coding sequence of a tedanalactamsynthase gene.

FIG. 9 represents a plasmid map of pMON25422 which contains a cDNAfragment representing a partial coding sequence of a tedanalactamsynthase gene.

FIG. 10 represents a plasmid map of pMON25424 which contains a fulllength cDNA representing the coding sequence of a tedanalactam synthasegene.

FIG. 11 represents a plasmid map of pMON25030 which represents a yeastexpression vector containing a DNA fragment encoding a lysine oxidase.

FIG. 12 represents a plasmid map of pMON25428 which contains a cDNAfragment encoding a tedanalactam synthase under the control of anarabinose inducible promoter.

FIG. 13 represents a plasmid map of pMON19469 which is a monocotexpression vector containing a cauliflower mosaic virus 35S promoter, anhsp70 intron, and a nopaline synthase polyadenylation sequence.

FIG. 14 represents a plasmid map of pMON25040 which contains a DNAsequence encoding a lysine oxidase variant under the control of acauliflower mosaic virus 35S promoter.

FIG. 15 represents a plasmid map of pMON15786 which is a plant transientexpression vector containing a neomycin phosphotransferase codingsequence fused to an hsp70 intron, under the control of a cauliflowermosaic virus 35S promoter.

FIG. 16 represents a plasmid map of pMON25041 which contains a DNAfragment encoding a lysine oxidase variant under the control of acauliflower mosaic virus 35S promoter.

FIG. 17 represents a plasmid map of pMON30411 which is a planttransformation vector containing a tedanalactam synthase variant geneunder the control of a cauliflower mosaic virus 35S promoter.

FIG. 18 represents a plasmid map of pMON30410 which is a planttransformation vector containing a tedanalactam synthase gene under thecontrol of a cauliflower mosaic virus 35S promoter.

FIG. 19 represents a plasmid map of pMON30417 which is a planttransformation vector containing a neomycin phosphotransferaseselectable marker, a tedanalactam synthase gene, and a lysine oxidasegene each under the control of a separate cauliflower mosaic virus 35Spromoter.

FIG. 20 represents a plasmid map of pMON25058 which is a plant transientis expression vector containing a lysine oxidase gene fused to an hsp70intron under the control of a root tissue enhanced promoter.

FIG. 21 represents a plasmid map of pMON25043 which is a plant transientexpression vector containing a tedanalactam synthase gene fused to anhsp70 leader under the control of a figwort mosaic virus promoter.

FIG. 22 represents a plasmid map of pMON10098 which is an Agrobacteriummediated double border plant transformation vector containing a neomycinphosphotransferase selectable marker under the control of a cauliflowermosaic virus 35S promoter.

FIG. 23 represents a plasmid map of pMON25046 which is an Agrobacteriummediated double border plant transformation vector containing a neomycinphosphotransferase selectable marker under the control of a cauliflowermosaic virus 35S promoter, and a tedanalactam synthase gene fused to anhsp70 leader under the control of a figwort mosaic virus promoter.

FIG. 24 represents a plasmid map of pMON25042 which contains a lysineoxidase gene fused to a petunia hsp70 leader sequence under the controlof a figwort mosaic virus promoter.

FIG. 25 represents a plasmid map of pMON25050 which is an Agrobacteriummediated double border plant transformation vector containing a neomycinphosphotransferase selectable marker under the control of a cauliflowermosaic virus 35S promoter, and a lysine oxidase gene fused to a petuniahsp70 leader sequence under the control of a figwort mosaic viruspromoter.

FIG. 26 represents a plasmid map of pMON33700 which is a plant transientexpression vector containing a lysine oxidase gene fused to a sequenceencoding a maize ATP synthase beta subunit mitochondrial transitpeptide, which is fused to an hsp70 intron sequence under the control ofa root tissue enhances promoter.

FIG. 27 represents a plasmid map of pMON33701 which is a planttransformation vector containing a neomycin phosphotransferase geneunder the control of a cauliflower mosaic virus 35S promoter, and atedanalactam synthase gene fused to an hsp70 intron sequence under thecontrol of a root tissue enhanced promoter.

FIG. 28 represents a plasmid map of pMON33702 which is a planttransformation vector containing a neomycin phosphotransferase geneunder the control of a cauliflower mosaic virus 35S promoter; a lysineoxidase gene fused to a sequence encoding a maize ATP synthase betasubunit mitochondrial transit peptide, which is fused to an hsp70 intronsequence under the control of a root tissue enhanced promoter; and atedanalactam synthase gene fused to an hsp70 intron sequence under thecontrol of a root tissue enhanced promoter.

FIG. 29 represents a plasmid map of pMON38800, which is a planttransformation vector containing a neomycin phosphotransferase geneunder the control of a cauliflower mosaic virus 35S promoter; a lysineoxidase gene fused to an amino terminal His6 coding region, a ricemalate dehydrogenase amino terminal peroxisomal targeting signal, anintron and a 5′ wheat chloroplast AB untranslated leader under thecontrol of a 4AS1 promoter and a wheat 17 kd heat shock protein 3′untranslated sequence; and a tedanalactam synthase gene fused to anintron under the control of a 4AS 1 promoter and a nopaline synthase 3′untranslated sequence.

DETAILED DESCRIPTION OF THE INVENTION

The following detailed description of the invention is provided to aidthose skilled in the art in practicing the present invention. Even so,the following detailed description should not be construed to undulylimit the present invention as modifications and variations in theembodiments discussed herein may be made by those of ordinary skill inthe art without departing from the spirit or scope of the presentinventive discovery.

The work described herein has identified compositions and methods ofexpressing an amino acid oxidase gene in combination with a second geneencoding a protein which, when provided in a composition with the aminoacid oxidase, confers plant resistance to coleopteran insects.Agronomic, horticultural, ornamental, and other economically orcommercially useful plants can be made in a functionally operablemanner, described herein, to express effective levels of protein toconfer resistance to coleopteran insects. Such plants may co-express thegenes encoding the amino acid oxidase and the second gene along withother genes encoding antifungal, antibacterial, or antiviralpathogenesis-related peptides, polypeptides, or proteins; insecticidalproteins; proteins conferring herbicide resistance; and genes encodingproteins involved in improving the quality of plant products oragronomic performance of plants. Simultaneous co-expression of multipleproteins in plants is advantageous in that it exploits more than onemode of action to control plant pathogenic damage. This can minimize thepossibility of developing resistant pathogen strains, broaden the scopeof resistance, and potentially result in a synergistic effect, therebyenhancing the level of resistance. Note WO 92/17591, for example, inthis regard. Examples are provided herein in which a lysine oxidase anda second gene which encodes a tedanalactam synthase are used incombination to control coleopteran insects through either direct feedingor by use of microbes expressing genes which encode these proteins.

As used herein, the term composition means a mixture of ingredientswhich, when combined, provides an insecticidally effective substancecontaining an amount of the active ingredients encoded by a first enzymecomprising an amino acid oxidase and a second enzyme that providesinsecticidal activity when the second enzyme is present in the mixturewith the first enzyme. The composition can be an artificial mixturecomprising the two active ingredients along with aqueous or non-aqueousingredients which may be combined to provide a substrate suitable forfeeding to an insect. The feeding insect can be a larvae form or may bean adult form. The composition can also be a mixture which contains oneor more bacteria expressing the genes encoding the first and the secondenzymes, such that the mixture, when provided in a suitable form forfeeding to insects, causes an insecticidally effective amount of the twogene products to be delivered to the feeding insects. The compositioncan also be a mixture within plant tissues or plant cells, produced as aresult of expression of the genes encoding the first and the secondenzymes by the plant nuclear or nucleolar genome, by the plantmitochondrial genome, or by the plant chloroplast genome, such that themixture, when consumed by a feeding insect, causes an insecticidallyeffective amount of the two gene products to be delivered to the feedinginsects.

By “controlling insect infestation” it is meant that the compositionupon which susceptible insects feed, usually a crop expressing thecontemplated genes, is capable of delivering an insecticidally effectiveamount of the amino acid oxidase and the second gene product to thefeeding insect so that insect growth is stunted, or slowed, or that theinsect dies without causing an unnecessary or unacceptable amount ofcrop damage.

“A plurality of cells” is intended to mean two or more cells, and thecells can be of any type which are capable of being transformed with thegenetic constructs described herein, such as bacterial cells, plantcells, yeast cells, insect cells, and fungal cells.

“An insecticidally effective amount” is intended to mean any amount ofany composition described herein capable of providing insecticidalactivity upon ingestion of the composition by a susceptible insect,wherein the composition contains at least an amino acid oxidase and asecond gene product that provides insecticidal activity when present ina mixture with the amino acid oxidase. The insecticidal activity isreadily observed upon ingestion by a susceptible insect. A susceptibleinsect which consumes an insecticidally effective amount will notcontinue to grow at the same rate as a control susceptible insect.

Stringent conditions as related to polynucleotide hybridization is wellknown in the art, and so one skilled in the art would know that a numberof factors are applicable, both in influencing the stability of hybridpolynucleotide molecules and in influencing the hybridization rate ofpolynucleotides. For example, factors which influence hybrid stabilityinclude ionic strength of any hybridization solution; the basecomposition of the probe and the target polynucleotides; destabilizingagents present in the hybridization solution such as formamide or urea;the presence or availability of mismatched base pairs; and the duplexlength of the probe or target. All of these factors also influence thehybridization rate in addition to the temperature selected forhybridization; the viscosity of the solution; the complexity of theprobe, meaning the presence of repetitive sequences which would tend toincrease the hybridization rate; the pH of the hybridization solution;and the base composition of the probe and target. One skilled in the artwould be able to determine the optimum conditions for establishingstringency as related to identifying a polynucleotide by usinghybridization under stringent conditions to a probe of known base paircomposition.

Trichoderma sp. genes that encode 1) lysine oxidase (E.C. 1.4.3.14)proenzyme and 2) a M_(r) 50,000 tedanalactam synthase have been isolatedand sequenced. These new genes or genes from other organisms known toproduce or capable of producing lysine or other amino acid oxidases orproproteins and a tedanalactam synthase may be inserted into expressiblecassettes which can then be placed into a transformation vector for usein transforming plant-colonizing microorganisms which, when applied toplants, express the genes producing these proteins, thereby providingcontrol of insects. Alternatively, genes which are also part ofexpression cassettes, and which function in plants to encode the subjectproteins may be inserted into plant transformation vectors for use intransforming the genome of a plant or plant subcellular organelle suchas a mitochondria or chloroplast. Such transformed plants are thusprovided with a capability to protect itself from attack by expressingthe recombinant gene or genes and producing a lysine or amino acidoxidase along with a tedanalactam synthase. Additionally, the plantexpressing this combination of insecticidal proteins may also betransformed or crossed with other recombinant plants to obtain plantsexpressing B.t. genes for the control of either the same or additionalinsects. Alternatively, the recombinant plants expressing thiscombination of insecticidal proteins may be crossed to other, morepreferable germ plasm for purposes of providing commercial productlines. Examples of plants transformed to express B.t. genes aredisclosed in European Patent Publication No. 0385 962 (Fischhoff andPerlak, 1990).

An expression cassette is intended to mean a DNA molecule comprising apromoter sequence which functions to cause the production of an RNAsequence from a DNA sequence containing an open reading frame encodingeither an amino acid oxidase, a second enzyme which providesinsecticidal activity when present in a mixture with an amino acidoxidase, or a chimeric gene open reading frame constructed from the inframe fusion of an amino acid oxidase and a second enzyme which providesinsecticidal activity when present in a mixture with an amino acidoxidase. The promoter sequence is required to be operably or operativelylinked to the DNA sequence containing one or more of the open readingframes. It is understood that the promoter chosen is functional in aparticular cell type. The cell type in which a promoter is functionalcan be any known in the art, in particular a bacterial or bacterialvirus promoter is known to be functional in various bacterial species; aplant or plant virus promoter is known to be functional in various plantspecies or in various plant cells, either tissue specific or non-tissuespecific. A promoter which is root enhanced or root specific or roottissue specific is one which provides expression preferentially in cellsderived from root tissues in a variety of plant types, and moreparticularly in corn, rice, wheat, soybean, canola, and cotton. By rootenhanced it is meant that the promoter has been isolated or geneticallyengineered to preferentially express or be more active in root tissuewhen compared to other plant tissue types such as leaves, fruit,flowers, and such. In eukaryotic cell types, and preferably in plantcell types, a particular expressible cassette includes a 3′non-translated DNA sequence which functions in plants to cause theaddition of a polyadenylated nucleotide sequence to the 3′ end of an RNAsequence generated from the function of an upstream operable promoterand gene sequence.

Expressible cassettes can also contain other elements in addition tothose described above. For example, a DNA sequence which specifies a 5′non-translated leader sequence can be present downstream of the promoterbut upstream of the translatable gene sequence. DNA which specifiesleader sequences are generally found in association with particularpromoter sequences, yet have been successfully utilized when associatedwith heterologous promoters and gene sequences. Such leader sequence canincrease expression levels of desired proteins when associated withparticular genes. Another DNA sequence which can increase expressionlevels of desired proteins when associated with particular genesspecifies a non-translated RNA sequence known as an intron. Introns arewell known in the art. Intron sequences are excisedpost-transcriptionally from the initial RNA transcript while stillwithin the boundaries of the nuclear envelope in a process known assplicing in which RNA 5′ adjacent to the intron sequence and 3′ adjacentto the intron sequence are fused to create the resulting MRNA sequencewhich is ultimately translated into a protein product. In thisapplication, intron sequences are preferred in gene constructs which areprepared for insertion into monocotyledenous species of plants, howeverthis is not meant to be limiting, because introns can function indicotyledenous species for the same purposes. Introns preferably areprovided downstream of a promoter but upstream of the gene codingsequence, however introns can also be placed within a gene codingsequence, or even downstream of a gene coding sequence but upstream of a3′ non-translated polyadenylation coding sequence. Other elements can besignal peptide encoding sequences or transit peptide encoding sequences.These are also well known in the art. Signal peptides are ubiquitous andare generally fifteen to thirty amino acids long and are directed totargeting a precursor peptide, often a nascent peptide, to a secretionor secretory apparatus within the cell. The secretory apparatus can befound on or within the cytoplasmic or intracytoplasmic membrane of abacterium or the endoplasmic reticulum, Golgi, or other vacuolar orcytoplasmic membrane surface to which the signal peptide directs theprecursor peptide. The signal peptide is generally cleaved by somefaction of the secretory apparatus to release a proenzyme in which casethe precursor would be a pre-proenzyme, or to release a mature peptide.A targeting or transit peptide encoding sequence similarly directs aprotein to a particular membrane surface, which can be either a plastid,a chloroplast, or a mitochondria. The targeting or transit peptide leadsthe attached protein sequence into the particular organelle, and isgenerally cleaved to release the mature peptide into the particularorganelle.

A DNA vector can be any of a number of constructions which are wellknown in the art. These can be selected from the group consisting of butnot limited to plasmid, bacmid, phage, cosmid, yeast artificialchromosome (YAC), bacterial artificial chromosome (BAC), plant virus, orlinear DNA or RNA, so long as the vector is capable of being deliveredto a target cell for the express purpose of either transforming the cellby incorporation into the genome of the cell, by stable or temporaryreplication or otherwise existing for a period of time within the targetcell so that the genes encoding proteins, which in combination provideor exhibit coleopteran insecticidal activity, are able to produce thecontemplated composition either for purposes of enzymatic orimmunological detection or for protection of a plant or plant cell frominsect damage.

The present invention includes not only the Trichoderma lysine oxidaseand tedanalactam synthase proteins, but also biologically equivalentpeptides, polypeptides, and proteins. The phrase “biologicallyequivalent peptides, polypeptides, and proteins” denotes peptides,polypeptides, and proteins that exhibit the same or similar activitieswhen assayed in comparison to the Trichoderma counterpart by in vitro orin vivo assays. The phrase “same or similar activities” denotes theability to perform the same or similar function as the Trichodermacounterpart. These peptides, polypeptides, and proteins can contain aregion or moiety exhibiting sequence similarity to a correspondingregion or moiety of the Trichoderma proteins disclosed herein, but thisis not required as long as they exhibit the same or similar activity astheir Trichoderma counterpart. Biologically equivalent peptides,polypeptides, and proteins may include, but are not limited to truncatedfragments deleted from the N-terminal end, C-terminal end, internalregions of the protein, or combinations thereof. Additionally, variantsresulting from changes in one or more amino acid to a different naturalor non-natural amino acid, deletions, or insertions of natural ornon-natural amino acids may result in a biologically equivalentcompound. Such variants may be naturally occurring materials, or may beproduced by mutagenesis or random mutagenesis of the encoding nucleotidesequence.

The present invention encompasses not only the Trichoderma DNA sequenceslisted, but also biologically functional equivalent nucleotidesequences. The phrase “biologically functional equivalent nucleotidesequences” denotes DNAs and RNAs, including genomic DNA, cDNA, syntheticDNA, and mRNA nucleotide sequences, that encode peptides, polypeptides,and proteins exhibiting the same or similar activities as theTrichoderma lysine oxidase or tedanalactam synthase when assayed by invitro or in vivo methods. Such biologically functional equivalentnucleotide sequences can encode peptides, polypeptides, or proteins thatcontain a region or moiety exhibiting sequence similarity to thecorresponding region or moiety of the Trichoderma counterpart.Nucleotide sequences may contain conservative amino acid changes,altering the codon usage for a particular amino acid, but leaving theencoded peptide, polypeptide, or protein sequence unchanged.Alternatively, nucleotide sequences may contain non-conservative changesincluding, but not limited to substitutions, deletions, additions, orcombinations thereof. These biologically functional equivalentnucleotide sequences may be naturally occurring or produced by in vitromethods. These biologically functional equivalent nucleotide sequencesare preferably 80% identical to their Trichoderma counterparts. Morepreferably, biologically functional equivalent nucleotide sequences are85%, 87.5%, 90%, 92.5%, 95%, 97.5%, and ideally 100% identical to theirTrichoderma counterparts. Biologically functional equivalent nucleotidesequences may be identified by their capability of hybridizing understringent conditions to the lysine oxidase or tedanalactam synthaseencoding sequences, or the complements thereof.

Methods for identifying other genes which encode a first gene encodingan amino acid oxidases and a second gene which encodes a protein thatprovides insecticidal activity when present in a mixture with an aminoacid oxidase are contemplated herein. Methods for identifying suchsequences are well known in the art, however the novelty of the secondgene provides a particular advantage to the invention. The second genedescribed herein as a tedanalactam synthase can be used to detect andidentify the presence of other genes of substantial similarity, meaninggenes which are capable of being detected by hybridization to thetedanalactam synthase gene. Any mixture or sample containing apolynucleotide sequence encoding a protein that provides insectidicalactivity when present in a mixture with an amino acid oxidase can beprobed with a labeled polynucleotide sequence which is or iscomplementary to all or a portion of the tedanalactam synthase gene. Theact of probing a sample with the tedanalactam synthase gene will providea probe/sample complex in mixtures which contain a homologous gene or aheterologous gene capable of binding to the probe. The probe/samplecomplex can be detected in any number of ways not limited to enhancedchemiluminescence, radioisotopic, fluorescent, or colorimetric methodswell known in the art. The complex can be isolated, particularly if themethod chosen has utilized a phage blot or a cell culture blot method.The polynucleotide or polynucleotides which bound to the probe can beisolated either from the probe/sample complex or derived from theparticular sample which gave rise to the probe/sample complex to yield agene which encodes a protein that provides insectidical activity whenpresent in a mixture with an amino acid oxidase. The gene isolated inthis way may or may not have tedanalactam synthase activity.

Antibodies can be generated to detect either of the active peptideswhich comprise the compositions herein. An amino acid oxidase enzyme ora tedanalactam synthase enzyme can be purified by any number of meanswell known in the art. Purified enzymes can be provided in adjuvant formfor injection into a variety of animals, also well known in the art.Preferably rabbits are used, however goats, guinea pigs, horses,turkeys, chickens, and even humans could be used for producing reagentgrade antiserum directed to particular epitopes of these proteins foruse in methods which utilize antibodies for detection and purificationof such peptides. While serum from animals provides polyclonalantibodies which are directed to a large number or a variety of epitopeson each protein, monoclonal antibodies could easily be produced bymethods well known in the art.

Kits could also be used, in particular when immunological reagents areavailable, such as antibodies directed to detection of amino acidoxidase or tedanalactam synthase enzyme, or by designing oligonucleotidesequences for use in detecting the presence of genes contemplated hereinby thermal amplification methods or in combination with immunologicalmethods.

The expression of a plant gene which exists in double-stranded DNA forminvolves transcription of messenger RNA (mRNA) from one strand of theDNA by RNA polymerase enzyme, and the subsequent processing of the mRNAprimary transcript inside the nucleus. This processing involves a 3′non-translated region which adds polyadenylate nucleotides to the 3′ endof the RNA. Transcription of DNA into mRNA is regulated by a region ofDNA usually referred to as the “promoter”. The promoter region containsa sequence of bases that signals RNA polymerase to associate with theDNA and to initiate the transcription of mRNA using one of the DNAstrands as a template to make a corresponding strand of RNA.

One skilled in the art will recognize that many promoters, 5′non-translated leader sequences, introns and polyadenylation sequencesmay be used in accordance with the present invention. Suitable examplesof promoter sequences include, but are not limited to nopaline synthase(NOS), octopine synthase (OCS), cauliflower mosaic virus 19S and 35S(CaMV19S, CaMV35S), ribulose 1,5-bisphosphate carboxylase (ssRUBISCO),figwort mosaic virus (FMV), asparagine synthase,glutathione-S-transferase, T-DNA, CPRFl, histone H3, wheat gliadin,nopaline synthase, Agrobacterium rhizogenes rolC, tobacco anionicperoxidase, napA storage protein, Cassava vein mosaic virus (CVMV),polyubiquitin, glycinin Gy2, mas, mustard CHS1, Chlorella virus adeninemethyltransferase, Arabidopsis phenylalanine ammonia-lyase, potato ubi3,and the tomato hmg2 promoter. Suitable examples of root enhanced or rootspecific promoter sequences include, but are not limited to CaMV derivedAS4, tobacco RB7, and the rice RC2 promoter. Suitable examples of intronsequences include, but are not limited to the maize heat shock protein70 (hsp70), rice actin, cox11, histone H3, RNA polymerase II,chloroplast DNA trnl, maize ADH1 intron 1, maize actin intron 3,Arabidopsis thaliana polyubiquitin, and the plant hemoglobin exon 2intron. Suitable examples of 5′ leader sequences include, but are notlimited to petunia heat shock protein 70 (hsp70), AMV RNA4, 16Sribosomal, Arabidopsis ACT2, Arabidopsis ACT8, TMV RNA, and soybean Gy2.Suitable examples of polyadenylation signals include, but are notlimited to Agrobacterium nopaline synthase (NOS) and the Pisum sativumRUBISCO small subunit E9.

A number of promoters which are active in plant cells have beendescribed in the literature. Such promoters may be obtained from plantsor plant viruses and include, but are not limited to, the nopalinesynthase (NOS) and octopine synthase (OCS) promoters (which are carriedon tumor-inducing plasmids of Agrobacterium tumefaciens), thecauliflower mosaic virus (CaMV) 19S and 35S promoters, thelight-inducible promoter from the small subunit of ribulose1,5-bisphosphate carboxylase (ssRUBISCO, a very abundant plantpolypeptide), and the Figwort Mosaic Virus (FMV) 35S promoter. All ofthese promoters have been used to create various types of DNA constructswhich have been expressed in plants (see e.g., Barry and Kishore, U.S.Pat. No. 5,463,175).

The particular promoter selected should be capable of causing sufficientexpression of the enzyme coding sequence to result in the production ofan insecticidal effective amount of lysine oxidase and tedanalactamsynthase. One set of preferred promoters are constitutive promoters suchas the CaMV35S or FMV35S promoters that yield high levels of expressionin most plant organs. Another set of preferred promoters are rootenhanced or specific promoters such as the CaMV derived AS4 promoter,the tobacco RB7 promoter, or the rice RC2 promoter (Lam et al., 1991;Yamamoto et al., 1991; Hertig et al., 1991; Xu et al., 1995). The rootenhanced or specific promoters would be particularly preferred for thecontrol of corn rootworm (Diabrotica spp.) in transgenic corn plants.

The promoters used in the DNA constructs (i.e. chimeric plant genes) ofthe present invention may be modified, if desired, to affect theircontrol characteristics. For example, the CaMV35S promoter may beligated to the portion of the ssRUBISCO gene that represses theexpression of ssRUBISCO in the absence of light, to create a promoterwhich is active in leaves but not in roots. The resulting chimericpromoter may be used as described herein. For purposes of thisdescription, the phrase “CaMV35S” promoter thus includes variations ofCaMV35S promoter, e.g., promoters derived by means of ligation withoperator regions, random or controlled mutagenesis, etcetera.Furthermore, the promoters may be altered to contain multiple “enhancersequence” to assist in elevating gene expression. Examples of suchenhancer sequences have been reported by Kay et al. (1987).

The RNA produced by a DNA construct of the present invention alsocontains a 5′ non-translated leader sequence. This sequence can bederived from the promoter selected to express the gene, and can bespecifically modified so as to increase translation of the MRNA. The 5′non-translated regions can also be obtained from viral RNAs, fromsuitable eucaryotic genes, or from a synthetic gene sequence. Thepresent invention is not limited to constructs wherein thenon-translated region is derived from the 5′ non-translated sequencethat accompanies the promoter sequence. As shown below, a plant geneleader sequence which is useful in the present invention is the petuniaheat shock protein 70 (hsp70) leader (Winter et al., 1988).

For optimized expression in monocotyledenous plants, an intron shouldalso be included in the DNA expression construct. This intron wouldtypically be placed near the 5′ end of the mRNA in untranslatedsequence. This intron could be obtained from, but not limited to, a setof introns consisting of the maize hsp70 intron (Brown and Santino U.S.Pat. No. 5,424,412; 1995)or the rice Act1 intron (McElroy et al., 1990).As shown below, the maize hsp70 intron is useful in the presentinvention.

As noted above, the 3′ non-translated region of the chimeric plant genesof the present invention contains a polyadenylation signal whichfunctions in plants to cause the addition of adenylate nucleotides tothe 3′ end of the RNA. Examples of preferred 3′ regions are (1) the 3′transcribed, non-translated regions containing the polyadenylate signalof Agrobacterium tumor-inducing (Ti) plasmid genes, such as the nopalinesynthase (NOS) gene and (2) plant genes such as the pea ssRUBISCO E9gene (Fischhoff et al., 1987).

A chimeric plant gene containing the structural coding sequences of thepresent invention can be inserted into the genome of a plant by anysuitable method. Suitable plant transformation vectors include thosederived from a Ti plasmid of Agrobacterium tumefaciens, as well as thosedisclosed, e.g., by Herrera-Estrella (1983), Bevan (1983), Klee (1985)and EPO publication 0 120 516 (Schilperoort et al.). In addition toplant transformation vectors derived from the Ti or root-inducing (Ri)plasmids of Agrobacterium, alternative methods can be used to insert theDNA constructs of this invention into plant cells. Such methods mayinvolve, for example, the use of liposomes, electroporation, chemicalsthat increase free DNA uptake, free DNA delivery via microprojectilebombardment, and transformation using viruses or pollen (Fromm et al.,1986; Armstrong et al., 1990; Fromm et al., 1990).

To identify a transgenic plant expressing lysine oxidase and/ortedanalactam synthase, it is necessary to screen the herbicide orantibiotic resistant transgenic, regenerated plants (R0 generation) forexpression of these genes. This can be accomplished by various methodswell known to those skilled in the art, including but not limited to: 1)obtaining small tissue samples from the transgenic R0 plant and directlyassaying the tissue for activity against susceptible insects in parallelwith tissue derived from a non-expressing, negative control plant. Forexample, R0 transgenic potato plants expressing lysine oxidase andtedanalactan synthase can be identified by assaying leaf tissue derivedfrom such plants for activity against CPB; 2) analysis of proteinextracts by enzyme linked immunoassays (ELISAs) or immunoblot assaysspecific for lysine oxidase and/or tedanalactam synthase (antibodiesuseful for such detection schemes are described in the examples) or 3)reverse transcriptase PCR (RT PCR) to identify events expressing thegene of interest.

The following examples describe preferred embodiments of the invention.Other embodiments within the scope of the claims herein will be apparentto one skilled in the art from consideration of the specification orpractice of the invention as disclosed herein. It is intended that thespecification, together with the examples, be considered exemplary only,with the scope and spirit of the invention being indicated by the claimswhich follow the examples. In the examples all percentages are given ona weight basis unless otherwise indicated.

EXAMPLES

The present invention can be better understood from the followingillustrative, non-limiting Examples. Effective control of coleopteraninsect pests is demonstrated when these proteins are obtained orexpressed within their natural source organism, in heterologousmicroorganisms, or in transgenic plants.

Example 1

This example illustrates the discovery and characterization ofinsecticidal activity from a Trichoderma species.

The culture filtrate from a Trichoderma species, Monsanto fungal isolate# F22844, was found to exhibit insecticidal activity in a southern cornrootworm bioassay. The proteinaceous nature of the active component wassuggested by characterization experiments which showed that first, thecorn rootworm activity was completely lost after heating and second,only components within the filtrate which were larger than 10 kDa insize maintained insecticidal bioactivity (Table 1). A qualitative visualassessment of the assay revealed that the >10 kDa sample severelystunted the surviving larvae and also had ovicidal effects in additionto the mortality noted in Table 1.

A sample of a >10 kDa preparation of F22844 was utilized indiet-preincubation studies to determine if the corn rootworm activitywas due to a modification of the artificial diet. It was demonstratedthat diet pre-incubated with F22844 retained full insecticidal activity,while similar samples subjected to heat treatment prior to larvaladdition exhibited reduced bioactivity (Table 1).

An important consideration in determining the utility of an insecticidallead is bioactivity in bioassays using plant tissue in the assay media.F22844 retained its insecticidal activity in assays with plant tissuewhen a >10 kDa preparation of F22844 was added to BMS corn callus.Significant mortality of corn rootworm larvae feeding on this materialwas seen in this bioassay (Table 1).

TABLE 1 % Mortality Sizing and heat lability study [>10kDa] 68 [<10kDa]0 [>10kDa] - Heated 100° C. 0 Diet pre-incubation study Un-incubated 94Incubated 94 Incubated/Heated 80° C. 19 Callus diet assay [>10 kDa] 94

Example 2

This example illustrated the identification and characterization ofproteins with insecticidal activity isolated from Trichodermafermentation extracts.

SDS-PAGE analysis of chromatography fractions generated during thepurification of the southern corn rootworm-active protein(s) from F22844showed that major proteins of M_(r) 56,000 and M_(r) 50,000 were presentin the corn rootworm-active fractions. These proteins were then purifiedby sizing and ion exchange chromatography. This protocol consistentlyyielded significantly purified proteins. The bioassay results obtainedwith purified proteins from two separate fermentations of F22844 areshown in Tables 2a and 2b. In both cases, no bioactivity was detectedwhen the proteins were assayed individually but significant stunting wasseen when the proteins were combined in assay. These results demonstratethat it is a combination of the two proteins that is responsible for thecorn rootworm activity in F22844.

TABLE 2a Mean larval weight Sample Conc. (μg/mL) (mg) ± (SEM) % StuntingAcetate buffer 0 1.14 ± (0.17) — M_(r) 56,000 10 0.91 ± (0.09) NSS M_(r)50,000 1.5 0.83 ± (0.05) NSS M_(r) 56,000 + 10 + 1.5 0.37 ± (0.07) 67M_(r) 50,000 NSS = not statistically significant

TABLE 2b Mean larval weight Sample Conc. (μg/mL) (mg) ± (SEM) % StuntingAcetate buffer — 1.29 ± (0.16) — M_(r) 56,000 2 1.63 ± (0.22) — M_(r)50,000 2 1.39 ± (0.22) — M_(r) 56,000 + 2 + 2 0.56 ± (0.22) 57 M_(r)50,000

Native-PAGE studies were effective in confirming that the M_(r) 56,000and M_(r) 50,000 proteins were responsible for the southern cornrootworm bioactivity of F22844. The chromatographically purified M_(r)56,000 and M_(r) 50,000 proteins were further purified by native PAGE.SDS-PAGE analysis demonstrated that the proteins were purified to singlebands. The individual purified proteins did not yield statisticallysignificant stunting of corn rootworm (Table 3). A sample containing acombination of the two proteins at concentrations of 9.7 μg/mL of M_(r)56,000 and 2.5 μg/mL of M_(r) 50,000 yielded 89% stunting of cornrootworm larvae (Table 3).

TABLE 3 Mean larval weight Sample Conc. (μg/mL) (mg) ± (SEM) % StuntingAcetate buffer 0   0.88 ± (0.11) — M_(r) 56,000 9.7 0.84 ± (0.06) NSSM_(r) 50,000 2.5 1.04 ± (0.11) NSS M_(r) 56,000 + 9.7 + 2.5 0.10 ±(0.01) 89 M_(r) 50,000

An SDS-PAGE gel of the proteins produced by Trichoderma F22844, isolatedas above, were blotted onto a PVDF membrane (Immobilon, Millipore,Bedford, Mass.) using the protocol of Matsudaira (Matsudaira, 1987). TheN-terminus was sequenced using automated Edman degradation chemistry. Agas phase sequencer (Applied Biosystems, Foster City, Calif.) was usedfor the degradation using the standard sequencer cycle. The respectivePTH-aa derivatives were identified by reverse phase HPLC analysis in anon-line fashion employing a PTH analyzer (Applied Biosystems, FosterCity, Calif.) fitted with a Brownlee 2.1 mm i.d. PTH-C18 column. Forinternal sequences, digestions were carried out on purified lysineoxidase and M_(r) 50,000 proteins from F22844 using trypsin(TPCK-treated, from Worthington Biochemicals Corp., Freehold, N.J.).Fragments were then purified by reverse phase HPLC and sequenced in anN-terminal fashion.

F22844 was identified taxonomically as a Trichoderma species. A searchin the CRC antibiotic database for proteins produced by Trichodermayielded L-lysine oxidase (LO), an M_(r) 56,000 protein identified as anantitumor agent (Kusakabe et al., 1979; 1980). The purified M_(r) 56,000protein from F22844 was identified as L-lysine oxidase by enzymaticassay (Gallo et al., 1981). We determined the lysine oxidase from F22844to have a K_(m) for lysine of 26 μM, a value very similar to thereported K_(m) for lysine of 40 μM for the T. viride lysine oxidase(Kusakabe et al., 1980). The substrate specificity of F22844 lysineoxidase was also determined (Table 4). The relative reaction rates arevery similar to the values in the literature for T viride lysine oxidase(Kusakabe et al., 1980).

TABLE 4 Relative activity (%) Substrate^(a) F22844^(a) T. viride ^(b)Lysine 100.0 100 Ornithine 10.8 18.2 Phenylalanine 6.6 8.3 Histidine 5.03.8 Arginine 2.6 6.1 ^(a)Relative activities of less than 1% weredetected with L-isomers of leucine, proline, threonine, tryptophan,serine, alanine, asparagine, valine, methionine, tyrosine, glutamicacid, glutamine, isoleucine, glycine, aspartic acid and cysteine. ^(b)T. viride data from Kusakabe et al., 1980. No activity against L-isomersof leucine, proline, threonine, tryptophan, serine, alanine, asparagine,valine, methionine, tyrosine, glutamic acid, glutamine, isoleucine,glycine, aspartic acid and cysteine was observed (Kusakabe, et al.,1980).

Data generated thus far indicate that the M_(r) 50,000 protein catalysesthe conversion of Δ¹-piperideine-2-carboxylate (P2C) to product I. Highresolution mass spectral data suggest that I is the 3,4-epoxide of2-piperidone, which has previously been identified as tedanalactam(Cronan and Cardellina, 1994). Hydrogen peroxide appears to be thebyproduct of the M_(r) 50,000 protein reaction. The formation ofhydrogen peroxide was monitored by a colorimetric assay (Gallo, 1981).P2C was prepared by two separate methods for the initial M_(r) 50,000protein assay. The two methods used for preparing P2C were

1) L-lysine with lysine oxidase and catalase; and

2) DL-pipecolic acid with D-amino acid oxidase and catalase.

P2C was isolated from the protein mixture by filtration using an Amiconfilter (10 kDa cutoff) before using in the M_(r) 50,000 protein assay.Pipecolic acid and lysine are not substrates of M_(r) 50,000 protein.

Note that in proposing an enzymatic activity for the M_(r) 50,000protein, we do not limit our claims. More specifically, we propose thatboth the M_(r) 50,000 protein and any substantially homologous proteinswill yield coleopteran insect control when combined with lysine oxidaseand other amino acid oxidases. Moreover, it is conceived that any enzymecapable of converting Δ¹-piperideine-2-carboxylate to tedanalactam wouldbe considered by one skilled in the art to have tedanalactam syntheticactivity, and would be characterized as a tedanalactam synthase enzyme.Any tedanalactam synthase enzyme capable of convertingΔ¹-piperideine-2-carboxylate to tedanalactam could be used incompositions with lysine oxidase or other amino acid oxidases asdescribed herein for controlling insect infestation of plants, and mayalso be effective in controlling insect infestation in general whensupplied in any form capable of being ingested by an insect. Genesencoding tedanalactam synthases may not necessarily be homologous togenes encoding such enzymes isolated from fuigal species such asTrichoderma. Other tedanalactam synthase genes may encode enzymes whichwould be substantially smaller than such enzymes observed fromTrichoderma. Conversely, it is entirely possible that still othertedanalactam synthase genes may encode enzymes which are substantiallylarger than such Trichoderma enzymes. Therefore, it is not desired thatthis invention be limited to enzymes approximately M_(r) 50,000 havingtedanalactam synthase activity.

Example 3

This example illustrates the bioactivity of lysine oxidase and thetedanalactam synthase derived from naturally occurring source organisms.

Lysine oxidase and tedanalactam synthase were purified from culturefiltrates of the native F22844 fungus. Both proteins were greater than90% pure by SDS-PAGE. Concentration response curves were run todetermine the efficacy of these purified proteins on neonate westerncorn rootworm, Diabrotica virgifera virgifera LeConte larvae. An LC₅₀below 2 ppm of each protein was demonstrated (Table 5).

TABLE 5 Sample Conc. (ppm) % Mortality % Stunting LO + M_(r) 50,000 20 +20 100 — 10 + 10 100 —  2 + 2 63 80 Control — 0  0

Western corn rootworm, Diabrotica virgifera virgifera LeConte, bioassayswere also conducted using second instar larvae with lysine oxidasepresent at 30 ppm and tedanalactam synthase present at 4 ppm. By day 10,the western corn rootworm larvae exposed to the active proteins had acorrected mortality of 81% and the surviving larvae actually lost weightover the course of the assay (Table 6).

TABLE 6 # # # # Larval Larv. Larval Larv. Larv. Larv. #Surv. Wts.Initial Wt. (mg)- Surv. Surv. Surv. Surv. (mg)- Day Day 1 ± Day Day DayDay Day 10 ± Sample 1 (SEM) 4 7 9 10 (SEM) Buffer 48 2.63 ± 46 44 43 438.48 ± control (0.18) (0.57) Lysine 48 2.63 ± 38 21 10  8 1.44 ±oxidase + (0.18) (0.10) M_(r) 50,000

Concentration response studies using chromatographically purifiedproteins were conducted. A concentration of 10 ppm of each protein(lysine oxidase+tedanalactam synthase) yielded 80% stunting of southerncorn rootworm while 2 ppm of each yielded 60% stunting (Table 7).

TABLE 7 Mean larval weight Sample Conc. (μg/mL) (SEM) % Stunting Acetatebuffer  0 1.00 (0.11) — Lysine oxidase +  2 + 2 0.40 (0.05) 60 M_(r)50,000 Lysine oxidase + 10 + 10 0.20 (0.05) 80 M_(r) 50,000

The purified proteins were also bioassayed in a BMS callus diet andbioactivity was retained in this assay (Table 8). These datademonstrated that the proteins were bioactive against southern cornrootworm in plant based bioassays in addition to artificial dietbioassays.

TABLE 8 Mean larval weight Sample Conc. (μg/mL) (mg) ± (SEM) % StuntingArtificial Diet Acetate buffer 0 1.15 ± (0.28) — Lysine oxidase + 17 + 20.35 ± (0.06) 70 M_(r) 50,000 BMS Callus Acetate buffer 0 0.27 ± (0.04)— Lysine oxidase + 17 + 2 0.13 ± (0.01) 53 M_(r) 50,000

Bioassays were done with purified M_(r) 56,000+M_(r) 50,000 proteinsfrom F22844 against two other coleopteran insects. Bioactivity wasdetected against Colorado potato beetle, Leptinotarsa decemlineata(Say), and boll weevil, Anthonomus grandis grandis Boheman.

TABLE 9 % Mean larval wt % Sample Surv/Init Mortality (mg) ± (SEM)Stunting Boll weevil Buffer control 30/32 — 11.05 ± (1.49)  — LO + 50 (2ppm each) 13/16 13 12.07 ± (2.05)  — LO + 50 (6 ppm each) 11/16 27 5.67± (2.10) 49 LO + 50 (18 ppm  6/16 60 1.42 ± (0.48) 87 each) CO. potatobeetle Buffer control 31/32 — 3.31 ± (0.33) — LO + 50 (2 ppm each) 15/16 3 2.83 ± (0.24) 15 LO + 50 (6 ppm each) 12/16 22 2.57 ± (0.48) 23 LO +50 (18 ppm  8/16 48 2.08 ± (0.32) 37 each)

Culture supernatants from four other Monsanto fungal isolates that hadcorn rootworm insecticidal activity were strongly positive for lysineoxidase enzymatic activity- F25528, F25634, F26040 and F25508. Theseleads were not characterized further. Culture supernatants from ATCCT.viride isolates #20536 and #20538 expressed lysine oxidase and anM_(r) 50,000 protein and were insecticidally active against southerncorn rootworm. Lysine oxidase from Trichoderma viride is availablecommercially from Sigma (St. Louis, Mo.). The enzyme was purified to asingle band from this preparation and bioassayed against southern cornrootworm. Bioassays were conducted to compare the concentrationresponses of the T. viride lysine oxidase (±M_(r) 50,000 protein fromF22844) with the lysine oxidase purified from F22844. The bioactivity ofthe T viride lysine oxidase agrees very well with the bioactivity oflysine oxidase from F22844 when bioassayed in the presence oftedanalactam synthase from F22844 (Table 10). Very similar stuntinglevels are seen at equivalent doses (compare Table 10 to Table 7). Inaddition, no bioactivity is seen with either lysine oxidase in theabsence of tedanalactam synthase from F22844.

TABLE 10 Mean larval weight Sample Conc. (ppm) (mg) ± (SEM) % StuntingBuffer control  0 0.77 ± (0.09) — T. viride LO 10 0.74 ± (0.07)  4* T.viride LO  2 0.73 ± (0.09)  5* T. viride LO + 10 + 10 0.21 ± (0.04) 73F22844 M_(r) 50,000 T. viride LO + 2 + 2 0.39 ± (0.05) 49 F22844 M_(r)50,000 *not statistically significant

Example 4

This example illustrates mode of action studies of the lysine oxidaseand tedanalactam synthase and effects which were observed on the cornroot worm midgut.

The effects of lysine oxidase and tedanalactam synthase proteins on themorphology and ultrastructure of the southern corn rootworm midgut wereinvestigated by light and electron microscopy. Light microscopy showedthat the midguts were intact with microvilli but there was marked apicalfolding of epithelium and basal infolding in treated individuals and anapparent loss of the basal regenerative cells. Several ultrastructuralchanges were evident by electron microscopy. The rough endoplasmicreticulum of the epithelial cells was dramatically reduced in treatedindividuals indicating a reduced potential for protein synthesis. Therewas an increased electron density (osmiophilia) of lateral plasmamembranes suggesting an abnormality in lipid metabolism. In the fatbody, lipid vesicles were significantly reduced in treated individuals.These micrographs showed that there are some definite cellular changesassociated with the treatment of corn rootworm with the purifiedproteins.

Example 5

This example describes the isolation and characterization of the genesencoding a lysine oxidase and a tedanalactam synthase.

The lysine oxidase and tedanalactam synthase genes were isolated fromone of the Trichoderma sp. microorganisms isolated in Ecuador and thesequences determined.

Peptide sequences from purified tryptic peptides from F22844 lysineoxidase were obtained and used to design degenerate oligonucleotides toclone the lysine oxidase gene. From peptide P1 (SEQ ID NO:1) was deducedthe antisense strand oligonucleotide N1 (SEQ ID NO:2), from peptide P2(SEQ ID NO:3) was deduced the antisense strand oligonucleotide N2 (SEQID NO:4) and sense strand oligonucleotides N3, N4, and N5 (SEQ IDNOS:5,6,7). From peptide P3 (SEQ ID NO:8) was derived the sense strandoligonucleotides N6 (SEQ ID NO:9) and N7 (SEQ ID NO:10).

P1 (SEQ ID NO:1): Asp Ala Pro Pro Gln Pro Pro Lys Glu Asp Glu Leu ValGlu Leu Ile Leu Gln Asn Leu Ala Arg

N1 (SEQ ID NO:2): TC(AG) TC(CT) TC(CT) TTI GGI GG(CT) TG

P2 (SEQ ID NO:3): Gly Leu Asn Leu His Pro Thr Gln Ala Asp Ala Ile Arg

N2 (SEQ ID NO:4): ATIGC(AG)TCIGC(CT)TGIGTIGG(AG)TG

N3 (SEQ ID NO:5): CA(CT)CCIACICA(AG)GCIGA(CT)GCIAT

N4 (SEQ ID NO:6): AA(CT)CTICA(CT)CCIACICA(AG)GC

N5 (SEQ ID NO:7): AA(CT) TT(AG) CA(CT) CCI ACI CA(AG) GC

P3 (SEQ ID NO:8): Lys Gln Gln Ala Phe Gly Tyr Tyr Lys

N6 (SEQ ID NO:9): AA(AG)CA(AG)CA(AG)GCITT(CT)GGITA

N7 (SEQ ID NO:10): CA(AG)GCITT(CT)GGITA(CT)TA(CT)AA

To obtain a partial cDNA clone, nested sense and antisense degenerateoligonucleotides to three internal tryptic peptide sequences from thepurified lysine oxidase were randomly combined in separate pair-wise PCRreactions with total cDNA from Trichoderma F22844 (Lee at al, 1988). Thecombination of sense strand oligonucleotides from peptide P3 (SEQ IDNO:8) and an antisense strand oligonucleotide from peptide P1 (SEQ IDNO:1) yield a PCR product of approximately 800 bp representing aninternal portion of the lysine oxidase cDNA. More specifically, sensestrand oligonucleotides N6 (SEQ ID NO:9) or N7 (SEQ ID NO:10) werecombined with the antisense strand oligonucleotide N1 (SEQ ID NO:2) in a50 μL PCR reaction containing 50 picomoles of each oligonucleotide, 1XTAQ buffer (Perkin-Elmer, Norwalk, Conn.), 1.5 mM MgCl₂, 660 μM dNTPs,and 0.5 units of Taq polymerase. The thermocycling profile was 1 cycleat 94° C. 5 minutes, 80° C. for 5 minutes in the absence of Taqpolymerase followed by addition of Taq. After Taq addition, there were 3cycles of 94° C. for 1 minute, 30 second ramp to 37° C. for 30 seconds,a 2.5 minute ramp to 72° C. for 2 minutes. This was followed by a PCRprofile of 37 cycles of 94° C. for 1 minute, 48° C. for 1 minute, 72° C.for 2 minutes and terminated with a final incubation at 74° C. for 4minutes.

To clone the internal lysine oxidase cDNA fragment, the 800 bp PCRfragment can then be ligated as a blunt ended fragment into a cloningvector such as pBSIIKS+ (Stratagene, La Jolla, Calif.) digested withSmaI to produce plasmid pMON23671 (FIG. 3). DNA sequencing of thisfragment reveals DNA sequences coding for peptide fragments P2 (SEQ IDNO:3) and P4 (SEQ ID NO:11) as well as portions of peptide fragments P1(SEQ ID NO:1) and P3 (SEQ ID NO:8), confirming the identity of the cloneas a lysine oxidase cDNA fragment (SEQ ID NO:12). Note that the firstresidue of the P3 (SEQ ID NO:8) was later found to be a leucine ratherthan a lysine upon review of both the DNA and protein sequencing data.The oligonucleotide N7 (SEQ ID NO:10) apparently still hybridized andfunctioned under the conditions described since only the first twonucleotides at its 5 prime end are mismatched.

It is useful to identify a complete cDNA sequence to determine if thegenomic DNA contains introns that disrupt the coding sequence. Suchintrons could inhibit expression of the cloned gene in heterologoussystems such as plants. To obtain the complete sequence of the lysineoxidase cDNA, the standard RACE (Rapid Amplification of cDNA Ends)procedure (Frohman et al., 1988) was used to extend the cDNA sequencefrom the internal core cDNA sequence present in pMON23671.

To recover the 3′ end, cDNA synthesized from poly A enriched F22844 withthe 3′ Race Adapter primer (Gibco-BRL, Gaithersburg, Md.) was PCRamplified with the Universal Amplification Primer (Gibco-BRL,Gaithersburg, Md.) and the lysine oxidase sense strand oligonucleotideN8 (SEQ ID NO:13). This PCR reaction yields a product of approximately700 bp that can be re-amplified with the internally nestedoligonucleotide is N9 (SEQ ID NO:14) to yield a PCR product ofapproximately 650 bp. The 650 bp product was subsequently subcloned as ablunt ended fragment into EcoRV digested pBSIIKS+ to yield pMON23683(FIG. 4). Subsequent sequencing of this cDNA clone displayeduninterrupted homology to the genomic lysine oxidase genomic DNAsequence (SEQ ID NO: 15).

N8 (SEQ ID NO:13): ACCTCTACGAACTTGCGTTTACC

N9 (SEQ ID NO:14): CAACTCGCATTGGATCGTTGGTG

To recover the 5′ end, two sets of 5′ RACE reactions were performed. Inthe first set, poly A enriched RNA from F22844 was reverse transcribedinto cDNA with oligonucleotide N 10 (SEQ ID NO:16) and dC tailed withTerminal Transferase (Gibco-BRL, Gaithersburg, Md.). This cDNA was thenamplified first with oligonucleotide N11 (SEQ ID NO:17) and the AnchorPrimer or AP (Gibco-BRL, Gaithersburg, Md.). This PCR reaction wassubsequently re-amplified with oligonucleotide N12 (SEQ ID NO:18) andthe Universal Amplification Primer or UAP (Gibco-BRL, Gaithersburg, Md.)to yield a 300 bp product which was blunt end cloned into EcoRV digestedpBSIIKS+ to yield pMON23681 (FIG. 5). Sequence analysis of this cDNAclone (SEQ ID NO:19) revealed uninterrupted homology to the genomiclysine oxidase genomic DNA sequence. However, a second set of 5′ RACEreactions was then needed to recover the remaining 5′ portion of thelysine oxidase cDNA sequence. This was accomplished by use of oligo dTprimed first strand cDNA as template, followed by one round of PCRamplification with the AP and N13 (SEQ ID NO:20), and completed with afinal round of PCR amplification using the preceding PCR reactionproduct as template with the UAP and N14 (SEQ ID NO:21) oligonucleotidesas primers. The final 520 bp, 5 prime RACE product was subcloned intothe PCR II vector (Invitrogen, San Diego, Calif.) to yield pMON25433 andsequenced to obtain the remaining cDNA sequence (SEQ ID NO:22). Thissequence also displayed uninterrupted homology to the lysine oxidasegenomic sequence.

N10 (SEQ ID NO:16) CAT GTC GTC GAC GAG CAT GAG C

N11 (SEQ ID NO:17) CAT CGA ACC CTT TGT CGA AGT CC

N12 (SEQ ID NO:18) CAG CAA GCT TCT CTT TGT AAT ACC C

N13 (SEQ ID NO:20) GTC GAA GTC CTC AGC CAG CTT CTC TTT GTA A

N14 (SEQ ID NO:21) CAT GCT GGG GAT GTC AGG

To obtain a complete clone of the genomic DNA encoding the lysineoxidase, a lambda phage genomic DNA library (Frischauf et al., 1987)derived from F22844 was screened by hybridization with the lysineoxidase partial cDNA fragment. In brief, approximately 20,000 plaqueforming units from the library were plated, transferred to filters, andprobed with radiolabelled lysine oxidase cDNA fragment (SEQ ID NO:12)which had been isolated from pMON23671. Briefly, filters were hybridizedwith a ³²P labeled probe (Feinberg and Vogelstein, 1983) with a specificactivity of approximately 2×10⁸ DPM/μg in 5×SSC, 5×Denhardts, 0.1% SDS,50% formamide, and 500 μg/mL DNA at 42° C. for 18 hours, washed twice in2×SSC, 0.1% SDS for 15 minutes at 25° C. or room temperature, washedtwice in 0.1×SSC, 0.1% SDS for 20 minutes at 60° C. andautoradiographed. Positive or hybridizing plaques were then picked,re-plated, and re-probed until a purified isolate consisting of only ofhybridizing plaques is obtained. Five independent, hybridizing lambdaphage clones were obtained.

The DNA from the purified lambda phage was then prepared by standardprocedures and analyzed by both direct DNA sequencing and southern blottechniques. Sequencing of the lambda genomic clones reveals essentiallycomplete sequence homology to the partial lysine oxidase cDNA clone inpMON23671 and to one another, indicating that the clones are independentisolates of the same gene. Southern blot analysis of lambda phagegenomic DNA digested with BamHI indicated that all of the clones carrieda common cross hybridizing BglII fragment of approximately 5 kb and thatgenomic DNA of the F22844 has a similar band. The 5 kb BglII fragmentfrom the lambda phage digest was subsequently isolated and cloned intothe BamHI site of pBSIIKS+(Stratagene, La Jolla, Calif.) to yieldpMON23680 (FIG. 6). An internal 1.0 kb XbaI fragment restriction mappedto the 3′ end of the genomic DNA was deleted from pMON23680 to yieldpMON23684 (FIG. 7). The complete sequence of the genomic DNA encodingthe lysine oxidase gene is given (SEQ ID NO:15).

The complete sequence of the genomic clone of the F22844 lysine oxidasegene was determined and compared to the sequence of the lysine oxidasecDNA. Comparison of the genomic and cDNA sequence indicates that thelysine oxidase gene has no introns within its coding region. Morespecifically, the DNA sequences of the genomic DNA (in lambda clone 56-3and derived pMONs 23680 and 23684) were determined by automatedsequencing of both strands (Prism DyeDeoxy Cycle Sequencing—AppliedBiosystems, Foster City, Calif.) and confirmed with manual sequencing(Sanger dideoxy chain termination). Sequences of the cDNA fragments inpMONs 23671, 23681, 23683 and pMON25433 were obtained by automatedsequencing as well as by manual sequencing for pMON25433.

Analysis of the lysine oxidase genomic sequence shows a single openreading frame encoding a 617 amino acid residue ORF of approximatepredicted M_(r) 69,400. Since the native protein has an apparent M_(r)56,000 this ORF encodes a pre-protein that is post-translationallymodified to yield the mature protein. N-terminal sequence data and massspectroscopy data indicate that approximately 77 amino acid residues arecleaved from the N-terminus to yield the mature lysine oxidase proteinof approximate M_(r) 60,000.

A search of the SWISS PROT database with the entire 617 amino acidresidue lysine oxidase ORF (SEQ ID NO:46; predicted M_(r)=69,400)encoded by the genomic sequence revealed homology of the Trichodermalysine oxidase to the Neurospora L-amino acid oxidase (LAO) precursorprotein (Niedermann and Lerch, 1990). The overall homology score was 24%identity, 50% similarity over 606 residues with the highest conservationin a region identified as a FAD binding site (8 of 9 contiguousresidues). Both lysine oxidase and LAO are apparently synthesized asproproteins since the first 129 amino acids of the non-secreted(intracellular) LAO proprotein are removed to yield the mature LAO(Niedermann and Lerch, 1990). This result suggests that the LAO ofNeurospora or other L-amino acid oxidases may be combined with theF22844 M_(r) 50,000 protein or other proteins to yield control of otherinsects.

The full length tedanalactam synthase cDNA was isolated in stages usingPCR based protocols of mixed oligonucleotide primed amplification ofcDNA (MOPAC) (Lee et al., 1988) and rapid amplification of cDNA ends(R.A.C.E.) (Frohman et al., 1988). First strand cDNA was generated frompoly A+ RNA isolated from 4 day old culture of F22844 and served astemplates for the MOPAC and RACE reactions. The first 5 prime (sense)gene specific amplification primer (GSP), primer N15 (SEQ ID NO:23), wasdesigned from the protein sequence P5 (SEQ ID NO:24) obtained fromtryptic peptide fragment 11. A second nested 5 prime GSP, primer N16(SEQ ID NO:25), was designed from protein sequence P6 (SEQ ID NO:26)derived from tryptic fragment 9. The 3 prime (antisense) GSP, primer N17(SEQ ID NO:27), was designed from the protein sequence P7 (SEQ ID NO:28)obtained from tryptic peptide fragment 16. A 623 bp partial cDNAfragment (SEQ ID NO:29) was obtained from an RT-PCR reaction withprimers N16 (SEQ ID NO:25) and N17 (SEQ ID NO:27), and subcloned intopBluescript II KS+ (Stratagene, La Jolla, Calif.) at the SmaI site(blunt ligation) resulting in pMON25421 (FIG. 8). The partial cDNA wassequenced and the deduced protein sequence matched the protein sequenceobtained from tryptic fragments 9, 7, and 16 (SEQ ID NO:26, SEQ IDNO:30,SEQ ID NO:28).

N15 (SEQ ID NO:23): GAR CAR AAY AAY TTY TTY AAY CAY GC

P5 (SEQ ID NO:24): Val Val Val Leu Glu Gln Asn Asn Phe Phe Asn His AlaGly Ser Ser Asn Asp Leu Ala

N16 (SEQ ID NO:25): ATG TAY ACI GAR CAY TAY ATG

P6 (SEQ ID NO:26): Thr Met Tyr Thr Glu Asp Tyr Met Ala Asp Leu Ala Lys

N17 (SEQ ID NO:27): GG IGC RAA YTG RAA CCA CAT

P7 (SEQ ID NO:28): Gly Thr Ile Phe Pro Ser Met Trp Phe Gln Phe Ala ProAsp Lys

P8 (SEQ ID NO:30): Leu Gly Met Thr Tyr Gln Glu Met Ser Ala Lys

RACE was used to identify the remaining 5 prime and 3 prime cDNAsequence of the tedanalactam synthase gene. 5 prime RACE was performedaccording the Gibco-BRL kit using P6 (SEQ ID NO:26) derived genespecific antisense primers N18 (SEQ ID NO:31) and N19 (SEQ ID NO:32).The 380 bp, 5 prime RACE product (SEQ ID NO:33) was subcloned into thePCR II vector (Invitrogen, San Diego, Calif.) resulting in pMON25422(FIG. 9). To recover the 3 prime portion of the cDNA, 3 prime RACE wasperformed using a P6 (SEQ ID NO:26) derived gene specific sense primerN20 (SEQ ID NO:34) and the Universal Amplification Primer (Gibco-BRL,Gaithersburg, Md.) which generated a 1423 base pair fragment. The 1423bp fragment 3 prime race product (SEQ ID NO:35) was subcloned into PCRII vector (Invitrogen, San Diego, Calif.) to yield pMON25423.

The full length 50 kb cDNA was generated by overlap PCR (Horton et al.,1989) using F22844 first strand cDNA as template and primers:

1) N21 (SEQ ID NO:36), a 5 prime sense primer that introduces BglII andNcoI restriction sites at the start codon (ATG) (primer mr50000-1)

2) N22 (SEQ ID NO:37) sense and N23 (SEQ ID NO:38) antisense 31-mers(primer mr50000-2 and mr50000-3) to remove the internal NcoI site.

3) N24 (SEQ ID NO:39) an oligonucleotide that introduces a EcoRI andHindIII restriction sites 3 prime to the stop codon that is located 1363bp downstream of the start codon (ATG).

The engineered full length cDNA, 1385 bp PCR product (SEQ ID NO: 40),was subcloned into PCR II vector (Invitrogen, San Diego, Calif.)resulting in pMON25424 (FIG. 10). The deduced translated proteinsequence is shown (SEQ ID:41).

N21 (SEQ ID NO:36): GGG AGA TCT CCA TGG CAG ACG AAA TCT

N22 (SEQ ID NO:37): GGC TTT CCA GCA CTT CCT TGG GGC CCT CCA A

N23 (SEQ ID NO:38): TTG GAG GGC CCC AAG GAA GTG CTG GAA AGC C

N24 (SEQ ID NO:39): CCC AAG CTT GAA TTC ACT TTC TTC TAT TGC C

Genomic DNA was isolated from the fungal pellet of a 5 day old liquidculture of F22844 (Fedoroff et al. 1983). Southern blot analysisindicated that the gene encoding tedanalactam synthase was a single copygene and mapped to an 8.0 kb BglII fragment. A F22844 genomic librarywas constructed from genomic DNA partially digested with MboI ligatedinto the BamHI site of the lambda EMBL3 vector (Frischauf et al., 1987).pMON 25421 cDNA insert was used to screen 48,000 plaque forming unitsfrom the primary library and nine positive overlapping clones wereidentified. The tedanalactam synthase gene was localized to a 9.0 kbSalI fragment. In three of the lambda clones, the gene mapped to one ofthe vector arms indicating that partial MboI digestions of F22844genomic DNA used in the construction of the library had createdtruncation of the 9.0 kb SalI fragment to a 6.0 kb, 4.4 kb and 2.5 kb.The 4.4 kb SalI fragment was subcloned into pBluescript II KS+(Stratagene, La Jolla, Calif.) resulting in pMON25425. The insert wassequenced and contained the complete 50 kb genomic clone with 5 introns(SEQ ID NO:42).

Example 6

This example illustrates bioactivity of lysine oxidase and tedanalactamsynthase derived from cloned genes against western corn rootworm.

The lysine oxidase and tedanalactam synthase genes can be isolated fromnovel sources or known sources. These genes may than be used totransform bacterial, yeast or plant cells, resulting in the productionof lysine oxidase (or lysine oxidase proprotein) and tedanalactamsynthase and permitting use of the methods of this invention. Examplesof how this could be done for the lysine oxidase and tedanalactamsynthase genes from fungal isolate F22844 are given below.

To introduce restriction endonuclease sites permitting expression of thelysine oxidase structural gene in transformed microorganisms and plants,the cloned genomic DNA sequence of the F22844 lysine oxidase gene (SEQID NO:15 in pMON23680 or 23684) was subjected to PCR mediatedsite-directed mutagenesis. Briefly, about 100 picograms of pMON23680 ina 100 μL reaction containing 1×Pfu buffer (Stratagene, La Jolla,calif.), 100 μM dNTPs, 0.5 μM oligonucleotide primer N25 (SEQ ID NO:43),0.5 μM oligonucleotide primer N26 (SEQ ID NO:44), 2.5 units of Pfupolymerase (Stratagene, La Jolla, Calif.) was overlaid with mineral oiland subjected to thermal cycling in a Perkin Elmer Model 480 DNA ThermoCycler. The thermal cycling profile was 1 cycle at 94° C. for 1.5minutes, 50° C. for 1 minute, 74° C. for 3 minutes followed by 25 cyclesof 94° C. for 1 minute, 50° C. for 1 minute, 74° C. for 2 minutes andterminated with final cycle of 94° C. for 1 minute, 50° C. for 1 minute,74° C. for 15 minutes. The PCR reaction product of approximately 1,900bp was electrophoresed on an agarose gel, purified (Qiagen, Chatsworth,Calif.), and digested with restriction endonucleases NcoI and EcoRI. Thesequence of the resultant PCR product is given (SEQ ID NO:45). Note thatthe amino acid sequence of the lysine oxidase M_(r) 69,400 proprotein(SEQ ID NO:46) encoded by the mutagenized DNA sequence is identical tothe deduced translation product of unmutagenized lysine oxidase genomicsequence (SEQ ID NO:15; translation start at base number 663,translational stop at base number 2513).

N25 (SEQ ID NO:43): TTGCAAACCATGGACAATGTTGACTTTGCTGAATC

N25 (SEQ ID NO:44): GCCGTAGTACCGAATTCTTATTAAATCTTCACC

Convenient restriction sites for placing the tedanalactam synthase geneinto plant expression vectors were also introduced by PCR site-directedmutagenesis as described previously.

To obtain functional lysine oxidase protein from the cloned gene, thelysine oxidase gene was engineered for expression in a heterologousyeast system. The yeast expression construct, pMON25030 (FIG. 11), wasmade by cloning a PCR generated fragment encoding the lysine oxidaseM_(r)69,400 proprotein (SEQ ID NO:46) into a pYES2 yeast expressionplasmid (Invitrogen, San Diego, Calif.). The PCR fragment (approximately1.9 kb) was generated by using two primers, N26 (SEQ ID NO:47) and N27(SEQ ID NO:48) and PCR amplifying a segment of DNA from pMON23680 (SEQID NO:15). Each primer carried a unique restriction site which provideddirectional cloning of the fragment into the pYES2 vector under GAL1promoter control. Primer N26 (SEQ ID NO:47) carried a BglII site whileN27 (SEQ ID NO:48) had an XbaI site. The resulting PCR fragment wasdigested with BglII and XbaI and cloned into BamHI and XbaI sites of thepYES2 vector. Note that the amino acid sequence of the lysine oxidaseM_(r) 69,400 proprotein (SEQ ID NO:46) encoded by this DNA fragment isnot affected by introduction of these restriction sites.

N26 (SEQ ID NO:47): CCCAGATCTATATTTGCAAACATGGACAATG

N27 (SEQ ID NO:48): GGGTCTAGACTAACAAACATCACACTTTCTATG

Yeast (Saccharomyces cerevisiae) transformed with pMON25030 were grownin the presence of galactose and shown to produce enzymatically activelysine oxidase. Yeast transformed with pMON25030 had soluble lysineoxidase activity in both disrupted cell pellets and cell free culturemedia. Culture media incubated for five days of galactose induction wasfound to hold the majority of lysine oxidase activity at approximately 1ng equivalent of lysine oxidase activity per 1 μl of media (activityunits are standardized to the activity of known amounts of F22844 lysineoxidase). Lysine oxidase from the yeast culture media was subsequentlypurified and shown to have bioactivity against corn rootworm whencombined with tedanalactam synthase. Surviving larvae exhibit 51%stunting with the combination sample of recombinant lysine oxidasepurified from yeast and the F22844 culture filtrate purifiedtedanalactam synthase (Table 11). No significant stunting was seen witheither protein individually. The yeast purified lysine oxidase had aspecific activity of 25 U/mg protein versus a specific activity of 37U/mg protein when the enzyme is purified from F22844.

TABLE 11 Treatment Concentration Mean larval wt. (16 wells/sample) (ppm)# of survivors (mg) (±SEM) Acetate buffer — 61 0.94 ± 0.08 Acetatebuffer — 45 0.84 ± 0.07 Lysine oxidase 14 38 0.76 ± 0.06 M_(r) 50,000 1043 0.80 ± 0.07 Lysine oxidase + 14 + 10 36 0.44 ± 0.05 M_(r) 50,000

An NcoI-HindIII fragment containing the full length tedanalactamsynthase cDNA gene (SEQ ID NO:40) from pMON25424 was interested intopMON6235, an E. coli expression vector containing an arabinose induciblepromoter and G10 leader sequence to produce pMON25428 (FIG. 12). Weaternblot analysis showed that the E. coli expressed protein was crossreactive to polyclonal antibodies raised against the F22844 purifiedtedanalactam synthase. The relative migration of the expressed proteinon SDS-PAGE was identical to the F22844 purified tedanalactam synthase.The recombinant tedanalactam synthase was purified in a 5 step procedurefrom E. coli expressing the tedanalactam synthase gene. Lysine oxidasewas purified from a commercial preparation of T. viride lysine oxidase.Both the lysine oxidase and tedanalactam synthase were >95% pure bySDS-PAGE. In bioassays against SCRW neonate larvae, >80% stunting ofsurvivors was seen with the combination sample of T. viride lysineoxidase and the E. coli produced F22844 tedanalactam synthase (Table12). The number of survivors was also dramatically reduced. Nosignificant mortality or stunting was seen with either proteinindividually. This demonstrates that the heterologously expressed orrecombinant tedanalactam synthase is insecticidally active against cornrootworm when assayed in the presence of lysine oxidase.

TABLE 12 Treatment (16 # of Mean larval wells/sample) Concentration(ppm) survivors wt. (mg) (+SEM) Tris/acetate buffer — 51 0.68 ± 0.04Tris/acetate buffer — 52 0.57 ± 0.04 Lysine oxidase 10 46 0.48 ± 0.04M_(r) 50,000 18 80 0.52 ± 0.03 Lysine oxidase + 10 + 18 23 0.11 ± 0.01M_(r) 50,000

Lysine oxidase isolated from yeast transformed with pMON25030 andtedanalactam synthase isolated from E. coli transformed with pMON25428were tested in bioassays for activity against western corn rootworm(Diabrotica) when combined respectively with tedanalactam synthase orlysine oxidase from F22844. Concentration response curves were run todetermine the efficacy of these purified proteins on neonate WCRW larvae(Table 13). Excellent bioactivity was observed with LC₅₀'s below 2 ppmfor each protein.

TABLE 13 Sample Conc. (ppm) % Mortality % Stunting F50/FLO¹ 20 + 20 100— 10 + 10 100 —  2 + 2 63 80 F50/YLO² 20 + 20 100 — 10 + 10 100 —  2 + 290 87 E50/FLO³ 10 + 10 84 80 ¹F22844 tedanalactam synthase and F22844lysine oxidase ²F22844 tedanalactam synthase and yeast lysine oxidase(recombinant) ³ E. coli tedanalactam synthase (recombinant) and F22844lysine oxidase

Example 7

This example illustrates the production of antibodies useful fordetection and quantitation of lysine oxidase and tedanalactam synthase.

To detect and quantitate tedanalactam synthase, polyclonal antibody #208was developed by immunizing a rabbit with partially purified 50K proteinderived from the E. coli expression vector pMON25428. A quantitativeEnzyme Linked Immunoassay (ELISA) where polyclonal antibody #208 is boththe coating and a Horse Radish Peroxidase (HRP) conjugated antibody wasdevised. The linear range of the tedanalactam synthase ELISA is betweenapproximately 2.5 to 40 ng . Antibody #208 is also used for 50K Westernblot analysis using the Enhanced Chemi-Luminescence (ECL) method ofAmersham (cat #RPN 2106). The Western blot procedure utilizes antibody#208 as the primary antibody and an Amersham goat anti-rabbit HRPconjugated secondary antibody (cat #NA 934). Incubation in the ECLreagents allows visualization of the antigens by autoradiography.

To detect and quantitate lysine oxidase, polyclonal antibody #450 wasdeveloped by immunizing a rabbit with partially purified lysine oxidaseprotein derived from the S. cerevisiae expression vector pMON25030. Aquantitative Enzyme Linked Immunoassay (ELISA) for lysine oxidase wherepolyclonal antibody #450 is both the coating and HRP-conjugated antibodywas devised. The linear range of the lysine oxidase ELISA is betweenapproximately 0.25 to 4.0 ng.

For analysis of the lysine oxidase by Western blot analysis, polyclonalantibody #2262 sera #3 was developed by immunizing a rabbit with a KLHconjugated 25-mer peptide CSVGEKLQQAFGYYKEKLAEDFDKG where all but theN-terminal cysteine residue is derived from the lysine oxidase peptidesequence. This antibody will detect both the lysine oxidase proenzyme ofapproximate M_(r) 69,000 as well as the enzymatically active and matureform of approximate M_(r) 69,000. Antibody #2262 sera #3 is used for LOWestern blot analysis using the Enhanced Chemi-Luminescence (ECL) methodof Amersham (cat #RPN 2106). The Western blot procedure utilizesantibody #2262 sera #3 as the primary antibody and an Amersham goatanti-rabbit HRP conjugate as the secondary antibody (cat #NA 934).Incubation in the ECL reagents allows visualization of the antigens byautoradiography.

Example 8

This example illustrates control of insects by expression of lysine andM_(r) 50,000 proteins in plant colonizing bacteria.

To control insects, it may be desirable to express lysine oxidase andtedanalactam synthase in plant colonizing bacteria, and then apply thisbacteria to the plant. As the insect feeds on the plant, it ingests atoxic dose of lysine oxidase and tedanalactam synthase produced by theplant colonizers. Plant colonizers can be either those that inhabit theplant surface, such as Pseudomonas or Agrobacterium species, orendophytes that inhabit the plant vasculature such as Clavibacterspecies. For surface colonizers, the lysine oxidase and tedanalactamsynthase genes may be inserted into a broad host range vector capable ofreplicating in these Gram-negative hosts. Examples of these such vectorsare pKT231 of the IncQ incompatibility group (Bagdasarian et al., 1981)or pVK100 of the IncP group (Knauf and Nester, 1982). For endophytes thetedanalactam synthase and lysine oxidase genes can be inserted into thechromosome by homologous recombination or by incorporation of the geneonto an appropriate transposon capable of chromosomal insertion in theseendophytic bacteria.

Example 9

This example illustrates control of coleopteran insects by expression oflysine oxidase and tedanalactam synthase in transgenic monocotlyedenousplants.

To place the lysine oxidase gene in a vector suitable for expression inmonocotyledonous plants (i.e. under control of the enhanced CauliflowerMosaic Virus 35S promoter and linker to the hsp70 intron followed by anopaline synthase polyadenylation site as in Brown and Santino, U.S.Pat. No. 5,424,412; 1995), the vector pMON19469 (FIG. 13) was digestedwith NcoI and EcoRI. The larger vector band of approximately 4.6 kb waselectrophoresed, purified, and ligated with T4 DNA ligase to theNcoI-EcoRI fragment of approximately 1.9 kb containing the lysineoxidase gene (SEQ ID NO:45). The ligation mix was transformed into E.coli strain XL-1 Blue (Stratagene, La Jolla, Calif.). Carbenicillinresistant colonies were recovered and plasmid DNA recovered by DNAminiprep procedures. This DNA was subjected to restriction endonucleaseanalysis with enzymes such as NcoI and EcoRI (together), NotI, and PstIto identify plasmid pMON25040, which contains the lysine oxidase codingsequence fused to the hsp70 intron under control of the enhanced CaMV35Spromoter (FIG. 14). Expression of functional lysine oxidase by pMON25040in corn protoplasts was confirmed by electroporation of pMON25040 DNAinto protoplasts followed by enzymatic assays of the plant protoplastsfor lysine oxidase activity.

To place the lysine oxidase gene in a vector suitable for recovery ofstably transformed and insect resistant plants, the 3.6 kb NotIrestriction fragment from pMON25040 containing the lysine oxidase codingsequence fused to the hsp70 intron under control of the enhanced CaMV35Spromoter was isolated by gel electrophoresis and purification. Thisfragment was ligated into NotI digested, alkaline phosphatase treatedpMON15786, a plant transient expression vector containing the neomycinphosphotransferase coding sequence fused to the hsp70 intron undercontrol of the enhanced CaMV35S promoter (FIG. 15). Kanamycin resistantcolonies were obtained by transformation of this ligation mix into E.coli XL-1 Blue (Stratagene, La Jolla, Calif.) and colonies containingpMON25041 (FIG. 16) identified by restriction endonuclease digestion ofplasmid miniprep DNAs. Restriction enzymes such as NotI, EcoRV, HindIII,NcoI, EcoRI, and BglII can be used to identify the appropriate clonescontaining the NotI fragment of pMON25040 in the NotI site of pMON15786(i.e. pMON25041) in the orientation such that both genes are in tandem(i.e. the 3′ end of the lysine oxidase expression cassette is linked tothe 5′ end of the nptII expression cassette). This vector can beintroduced into the genomic DNA of corn embryos by particle gunbombardment followed by paromomycin selection to obtain corn plantsexpressing the lysine oxidase gene essentially as described in Brown andSantino U.S. Pat. No. 5,424,412. These plants can then be “crossed” bypollen transfer to plants containing the tedanalactam synthase gene(construction described below, from pMON30411) to obtain plants that areresistant to insect infestation, particularly corn rootworm (Diabroticaspp.). Alternatively, pMON30411 and 25040 could be co-bombarded toobtain plants that are resistant to insect infestation, particularlycorn rootworm (Diabrotica spp.).

To place the tedanalactam synthase gene in a vector suitable forexpression in monocotyledonous plants (i.e. under control of theenhanced Cauliflower Mosaic Virus 35S promoter and linked to the hsp70intron followed by a nopaline synthase polyadenylation site), the vectorpMON19469 was digested with NcoI and EcoRI. The larger vector band ofapproximately 4.6 kb was electrophoresed, purified, and ligated with T4DNA ligase to the NcoI-EcoRI fragment of approximately 1.4 kb containingthe tedanalactam synthase gene (SEQ ID NO: 40) obtained from the E. coliexpression vector pMON25428. The ligation mix was transformed into E.coli strain XL-1 Blue (Stratagene, La Jolla, Calif.), carbenicillinresistant colonies recovered and plasmid DNA recovered by DNA miniprepprocedures. This DNA was subjected to restriction endonuclease analysiswith NcoI and EcoRI, NotI, and SacI to identify clones containingpMON30410, containing the tedanalactam synthase coding sequence fused tothe hsp70 intron under control of the enhanced CaMV35S promoter (FIG.18). Expression of the tedanalactam synthase gene by pMON30410 in cornprotoplasts was confirmed by electroporation of pMON30410 DNA intoprotoplasts followed by Western blot analysis of the plant protoplastextracts for tedanalactam synthase cross reacting species of the sameM_(r) as native tedanalactam synthase.

To place the tedanalactam synthase gene in a vector suitable forrecovery of stably transformed and insect resistant monocot plants, the3.1 kb NotI restriction fragment from pMON30410 containing thetedanalactam synthase coding sequence fused to the hsp70 intron undercontrol of the enhanced CaMV35S promoter was isolated by gelelectrophoresis and purification. This fragment was ligated withpMON15786 treated with NotI and calf intestinal alkaline phosphatase(pMON15786 contains the neomycin phosphotransferase coding sequencefused to the hsp70 intron under control of the enhanced CaMV35Spromoter). Kanamycin resistant colonies were obtained by transformationof this ligation mix into E. coli XL-1 Blue (Stratagene, La Jolla,Calif.) and colonies containing pMON30411 (FIG. 17) identified byrestriction endonuclease digestion of plasmid miniprep DNAs. Restrictionenzymes such as NotI, EcoRV, HindIII, NcoI, EcoRI, and BglII may be usedto identify the appropriate clones containing the NotI fragment ofpMON30411 in the NotI site of pMON15786 (i.e. pMON30411) in theorientation such that both genes are in tandem (i.e. the 3′ end of thetedanalactam synthase expression cassette is linked to the 5′ end of thenptII expression cassette). This vector can be introduced into thegenomic DNA of corn embryos by particle gun bombardment followed byparomomycin selection to obtain corn plants expressing the tedanalactamsynthase gene. These plants can then be “crossed” by pollen transfer toplants containing the lysine oxidase gene (pMON25041; see descriptionbelow) to obtain plants that are resistant to insect infestation,particularly corn rootworm (Diabrotica spp.). Alternatively, pMON30411and 25040 could be co-bombarded to obtain corn plants that are resistantto insect infestation, particularly corn rootworm (Diabrotica spp.).

To place both the tedanalactam synthase and lysine oxidase genes in avector suitable for recovery of stably transformed and insect resistantplants, the 3.6 kb NotI restriction fragment from pMON25040 containingthe lysine oxidase coding sequence fused to the hsp70 intron undercontrol of the enhanced CaMV35S promoter was isolated by gelelectrophoresis and purification. This fragment was ligated withpMON30411 that was partially digested with NotI to obtain pMON30411plasmid DNA cut only once with NotI and treated with calf intestinalalkaline phosphatase (pMON30411 contains the neomycin phosphotransferasecoding sequence fused to the hsp70 intron under control of the enhancedCaMV35S promoter and the tedanalactam synthase coding sequence fused tothe hsp70 intron under control of the enhanced CaMV35S promoter).Partial digestion and isolation of pMON30411 plasmid with only one NotIsite cut was accomplished by digesting 5 μg of DNA in 1×HSB buffer(Boehringer-Mannheim, Indianapolis, Ind.), 10 μg/mL bovine serumalbumin, 10 μg/mL ethidium bromide and 0.4 units of NotI restrictionendonuclease followed by gel electrophoresis and purification of thelinearized plasmid DNA. Kanamycin resistant colonies were obtained bytransformation of this ligation mix into E. coli XL-1 Blue (Stratagene,La Jolla, Calif.). Colonies containing pMON30417 (FIG. 19) wereidentified by restriction endonuclease digestion of plasmid miniprepDNAs. Restriction enzymes such as NotI, SacI, HindIII BamHI, NcoI/EcoRI,EcoRV and BglII may be used to identify the appropriate clonescontaining the NotI fragment of pMON25040 in the NotI site of pMON30411upstream of the tedanalactam synthase gene (i.e. pMON30417) in theorientation such that all genes are in tandem (i.e. the 3′ end of thelysine oxidase cassette is linked to the 5′ end of the tedanalactamsynthase expression cassette and the 3′ end of the M_(r) 50,000expression cassette is linked to the 5′ end of the nptII expressioncassette). Expression of both the lysine oxidase and tedanalactamsynthase in genes in corn leaf protoplasts electroporated with pMON30417was observed. This vector can be introduced into the genomic DNA of cornembryos by particle gun bombardment followed by paromomycin selection toobtain corn plants expressing both the lysine oxidase and tedanalactamsynthase genes essentially as described in Brown and Santino U.S. Pat.No. 5,424,412. Transgenic corn plants expressing the lysine oxidase andtedanalactam synthase genes can be identified by an ELISA assay specificfor the two proteins or via an enzymatic assay for lysine oxidaseactivity. These plants may be resistant to insect infestation,particularly corn rootworm (Diabrotica spp.).

Both the lysine oxidase gene in pMON25040 and the tedanalactam synthasegene in pMON30410 were shown to express the respective F22844 genes incorn leaf protoplasts. Corn leaf protoplasts were electroporated withpMONs 25040 and 30410 as well as a pMON19649 control and incubated forabout 24 hours. Total protein was extracted and assayed for the presenceof enzymatic lysine oxidase activity (Table 14) or tedanalactam synthasecross reacting material by Western blot analysis. Both genes areexpressed in corn cells.

TABLE 14 ng LO activity/mg total protein¹ Vector cell pellet² culturemedia (conc)³ pMON25040 148 ± 12 411 ± 3 pMON19649 0 0 ¹Lysine oxidaseactivity equivalents in nanograms per mg of total extracted protein.Lysine activity equivalents were determined by assaying in parallel adilution series of known amounts of purified F22844 lysine oxidase.²cell pellet was extracted with glass beads and desalted prior to assay.³culture media was concentrated over a Centricon 10 column (Amicon).

Corn plants transformed with the monocot transformation vectorcontaining both the lysine oxidase and tedanalactam synthase expressioncassettes (i.e. pMON30417) were obtained as described above. Atransgenic event that expressed both lysine oxidase and tedanalactamsynthase was identified and outcrossed to yield progeny that expressboth the lysine oxidase and tedanalactam synthase proteins atapproximately 2.5 and 4.5 PPM, respectively (leaf expression levels asdetermined by an ELISA). Progeny plants of the pMON30417 event andgenotypically identical controls that do not express the lysine oxidaseor tedanalactam synthase proteins were infested with western cornrootworm eggs and scored for root damage by the Iowa Rating system(Hills and Peters, 1971) after 3 weeks of feeding. These results aresummarized in Table 15.

TABLE 15 pMON30417 mediated control of western corn rootworm intransgenic corn Treatment N¹ Mean RDR² (SE)³ Range⁴ pMON30417 10 3.0 (0)3 Control 5 4.6 (0.24) 4-5 ¹N is number of plants assayed. ²RDR is theRoot Damage Rating; 1-5 scale, 5 most severe. ³X2 = 19.095, P < 0.002⁴Range of values obtained.

In addition to the quantitative Root Damage Rating data demonstratingprotection of corn plant roots expressing the lysine oxidase andtedanalactam synthase proteins (pMON30417), a number of qualitativeobservations also indicate that these genes confer corn rootwormcontrol. First, the above ground nodal roots were intact in thepMON30417 plants but were destroyed in the control plants. Second, thecontrol plants had copious numbers of larvae within the stalk at thetime of examination while the pMON30417 plants had none.

A bare root assay was performed on segregating ‘Laffite’ plants tomeasure plant insecticidal activity and larval growth. Survival on thecontrol was 39% as compared to 4% on F22844. Those surviving on F22844were in very poor condition (Table 16). Because of the poor condition ofthe F22844 larvae, no larval weights were recovered.

TABLE 16 Percent survival and mean larval weight of WCR in a bare rootassay. Treatment N Survival (%) Larval weight (mg) Control 15 38.7 0.24F22844 9 4.4 —

The western corn rootworm whole plant bioassay was repeated with‘Laffite’ in ten inch pots. The larger pot would allow for near normalplant development, thus allowing for both the opportunity to studyinsect control and the phytotoxic effects. Positive plants (2-4 ppm)were approximately 30-40 percent stunted at the end of the assay periodfor the H99 and B73 pedigrees, respectively. The root system on thepositive plants appeared normal for that size of a plant. The insectcontrol on the positive plants appeared normal for that size of a plant.

The average root damage ratings of the F22844 plants were significantlylower than the negative plants, 2.6 and 2.7 versus 5.2 and 5.9 for theH99 and B73 pedigrees, respectively. The negative segregants showedsevere rootworm damage with 2-3 entire nodes of the roots pruned (Table17). Note that constitutive expression of enzymatically active LO canresult in plant height reductions (Table 17). Combinations of rootspecific promoters, LO69 proenzyme variants resistant to plant proteaseactivation, and/or organellar or extracellular targeting of LO69 or LO69proenzyme variants are anticipated to relieve this effect of LOexpression in plants.

TABLE 17 Mean root ratings (RDR) for F22844, Event ‘Laffite’, for both aB73 and H99 S1 cross from the R0 in a ten inch pot WCR bioassay PlantHeight Treatment Pedigree N RDR Mean RDR Range Mean (inch) F22844 H99 162.6 1-3 28.0 B73 10 2.7 1-3 30.6 Control H99 10 5.2 3-5 39.3 B73 8 5.95-6 49.6

To reduce plant height reductions caused by lysine oxidase expressionand to target expression of the insecticidal proteins to the rootsattacked by Diabrotica sp, promoters that limit expression of lysineoxidase to roots are valuable. In this example, the 4 as-1 root enhancedpromoter (Lam et al., U.S. Pat. No. 5,023,179; designated pAS4 inplasmid maps) was fused to a transcription unit containing the maizehsp70 intron, the lysine oxidase gene, and the nos polyadenylation sitein pMON25058 (FIG. 20). The 4 as-1 promoter was also fused to atranscription unit containing the maize hsp70 intron, the tedanalactamsynthase gene, and the nos polyadenylation site in pMON25060. Both thelysine oxidase and tedanalactam synthase transcription units weresubsequently combined in a vector containing a neomycinphosphotransferase coding sequence fused to the hsp70 intron undercontrol of the enhanced CaMV35S promoter to yield pMON25061 (FIG. 21).Transgenic corn plants expressing both lysine oxidase and tedanalactamsynthase genes were recovered as previously described.

One illustrative pMON25061 event, R44482, was further analyzed and shownto express lysine oxidase and tedanalactam synthase at relatively highlevels in root tissue and at relatively low levels in leaf tissue (2.9ppm lysine oxidase in root versus 0.9 ppm in leaf; 8.4 ppm tedanalactamsynthase in root versus 1.9 ppm in leaf). Event 44482 was also displayedsignificant levels of CRW resistance in both growth chamber and fieldtests (Table 18). Although plant height reductions associated with thepMON25061 transgene in R44482 were not eliminated, the level of stuntungis clearly less than that observed when Lysine oxidase is expressed froma constitutive CaMV e35S promoter (Tables 17 and 18).

TABLE 18 Corn Rootworm Damage Rating and Height of CRW infested andnon-infested pMON25061 plants and wild type controls CRW RDR¹ CRW RDR¹CRW RDR¹ (Height) [Growth (Height) [Field Test (Height) [Field PlantsChamber] Site #1] Test Site #2] 25061#44482  3.2 RDR  5.8 RDR  5.6 RDR(infested) (51.5 in) (47 in) (37 in) wild type 10.4 RDR 9.4 RDR 11.9 RDR(infested) (50.6 in) (60 in) (51 in) 25061#44482 ND ND ND (uninfested)(79 in) (84 in) wild type ND ND ND (uninfested) (84 in) (86 in) ¹RDR isthe Root Damage Rating; 1-15 scale, 15 most severe.

Example 10

This example illustrates construction of lysine oxidase and tedanalactamsynthase dicot plant expression vectors, production of dicot plantsexpressing these genes, and use of these plants to control insect pests.

To control coleopteran insect pests such as the Colorado potato beetle,Leptinotarsa decemlineata (Say), in dicotyledonous plants such aspotato, Solanum tuberosum, or to control boll weevil, Anthonomusgrandis, in cotton, Gossypium hirsutum, it would be useful to engineerthe lysine oxidase and tedanalactam synthase genes for expression indicotyledonous plants.

To express tedanalactam synthase in dicots, the HindIII/NcoI fragmentcontaining a duplicated or enhanced version of the FMV promoter (Rogers,1995) and petunia hsp70 5 prime untranslated leader (Winter et al.,1988) from pMON18411 was ligated into the NcoI/HindIII sites ofpMON30410 located 5 prime to the tedanalactam synthase coding region(SEQ ID NO: 40) to yield pMON25043, containing a tedanalactam synthasevariant under the control of an enhanced FMV promoter/petunia hsp70untranslated leader with a NOS polyadenylation signal (FIG. 21). Thecomposite eFMV/hsp70/tedanalactam synthase/nos gene cassette wasisolated as a HindIII/NotI fragment from pMON25043 and ligated into theHindIII and NotI sites in pMON10098, a double border planttransformation vector(FIG. 22), to form pMON25046 (FIG. 23). ThepMON25046 plasmid is an Agrobacterium-based plant transformation vectorthat places the composite eFMV/hsp70/tedanalactam synthase/nos gene andenhanced CaMV35S/neomycin phosphotransferase/nos kanamycin selectablemarker gene between the right and left T-DNA border fragments. Thisvector can be mobilized into Agrobacterium (Ditta et al., 1980) and usedto obtain transgenic dicotyledonous plants as described (Horsch et al.,1985).

To express lysine oxidase in dicots, the NcoI/SmaI fragment of thelysine oxidase gene from pMON25040 (SEQ ID NO: 45) was ligated into theNcoI and SmaI sites located between the enhanced FMV promoter/petuniahsp70 5 prime untranslated leader (utl or UTR) and the NOSpolyadenylation signal to yield pMON25042, containing a lysine oxidasevariant under the control of the enhanced FMV promoter/petunia hsp70leader with a NOS polyadenylation signal (FIG. 24). The compositeeFMV/hsp70 utl/lysine oxidase/nos gene from pMON25042 was isolated frompMON25042 on a single NotI fragment and subsequently ligated into theNotI site of pMON25048, a derivative of pMON10098, containing a singleNotI site and kanamycin selectable marker gene located between the rightand left T-DNA border sequences, to obtain pMON25050 (FIG. 25).pMON25050 can be mobilized into Agrobacterium (Ditta et al., 1980) andused to obtain transgenic dicotyledonous plants as described previously(Horsch et al., 1985).

To place both the tedanalactam synthase and lysine oxidase genes in avector suitable for recovery of stably transformed and insect resistantdicotyledonous plants, the NotI fragment containing the lysine oxidaseexpression cassette from pMON25042 was ligated into a single NotI sitein pMON25046 introduced by single hit partial NotI digestion to obtainpMON25049 (FIG. 2). The pMON25049 plasmid is an Agrobacterium mediatedtransformation vector containing a kanamycin selectable marker gene, thetedanalactam synthase expression cassette, and the lysine oxidaseexpression cassette between the right and left T-DNA border sequences.This vector can be used to obtain dicotyledonous plants expressing boththe tedanalactam synthase and lysine oxidase genes via the previouslydescribed Agrobacterium-mediated plant transformation techniques.

Arabidopsis plants were transformed with pMON25046 and pMON25050 via theAgrobacterium mediated meristem infiltration technique (Bechtold et al.,1993) and screened for expression of the tedanalactam synthase andlysine oxidase genes, respectively. Identification of tedanalactamsynthase expressors was via an ELISA assay specific for the tedanalactamsynthase protein. Lysine oxidase expressors were identified via directenzymatic assays. Pollen was transferred from lysine oxidase expressingpMON25050 lines to tedanalactam synthase expressing pMON25046 lines toobtain F1 progeny that express both the tedanalactam synthase and lysineoxidase genes. These F1 progeny plants that expressed both the lysineoxidase and tedanalactam synthase genes and appropriate controls wereexposed to southern corn rootworm larvae to determine if these geneswould cause larval stunting (as assayed by reductions in larval weightpost-feeding) and reduced feeding (as assayed by a Leaf Damage Rating).The results displayed in Table 19 demonstrate that these genes causesignificant larval stunting and yield reduced leaf damage when expressedin transgenic Arabidopsis plants.

TABLE 19 Activity of F22844 expressing Arabidopsis on SCRW as shown bymean larval weight (mg) and Leaf Damage Rating (LDR). N is the number oflarvae weighed. Treatment N Mean Larval wt. (mg) (±SEM) Mean LDR LO +M_(r) 50,000 25 0.23 (±0.02) 0.84 untransformed 49 0.44 (±0.02) 2.79 LOonly 25 0.47 (±0.03) 2.11 M_(r) 50,000 only 37 0.39 (±0.02) 2.00

It may also be advantageous to localize the lysine oxidase ortedanalactam synthase to the plastids. Proteins can be directed to thechloroplast by including at their N-termini a chloroplast transitpeptide (CTP). One CTP that has worked to localize heterologous proteinsto the chloroplast of dicotyledonous plants is from the RUBISCO smallsubunit gene of Arabidopsis, denoted ats1A. A variant of this transitpeptide that encodes the transit peptide, 23 amino acids of matureRUBISCO sequence, plus a reiteration of the transit peptide cleavagesite has been constructed for the successful chloroplast localization ofthe B.t.k. protein (Fischhoff and Perlak, 1990). It is anticipated thatthis same fragment of the atsla gene, when fused in frame to theamino-terminal coding region of the lysine oxidase or tedanalactamsynthase genes, will similarly result in chloroplast localization ofthese proteins in dicotyledonous plants. Note that this chloroplasttargeted expression cassette is subsequently cloned into a binaryAgrobacterium transformation vector, mobilized into disarmedAgrobacterium hosts and used to transform dicots.

To localize lysine oxidase in corn plastids, A fragment of the maizeRUBISCO small subunit gene denoted mSSU CTP or zmS1 containing aduplicated cleavage site (Russell et al., 1993) was used to localizeeither the lysine oxidase or tedanalactam synthase in chloroplasts ofcorn plants. For example, an XbaI/NcoI fragment (SEQ ID NO:49) encodingthe mSSU CTP derived from pMON22089 was ligated into the XbaI and NcoIsites of pMON30405 (lysine oxidase; SEQ ID NO:50) or pMON30410 (M_(r)50,000 protein) that are located between the hsp70 intron and codingregions of these genes to obtain in-frame fusions of the mSSU CTP to theN-termini of these proteins. The pMON30405 lysine oxidase sequence (SEQID NO:50) was obtained by PCR mutagenesis using pMON23684 (SEQ ID NO:15)as template with oligonucleotides N28 (SEQ ID NO:51) and N29 (SEQ IDNO:52) as primers. The mSSU CTP fusions to lysine oxidase or M_(r)50,000 protein under control of the enhanced CaMV35S promoter and thehsp70 intron are cloned as NotI fragments into pMON15786, a monocottransformation vector described above. Transgenic corn plants expressinglysine oxidase were obtained at a lower frequency when the plastidtargeting signal was employed, indicating that plastid targeting is nota preferred method of expressing lysine oxidase in transgenic corn.

Example 11

This example illustrates mitochondrial targeting of the tedanalactamsynthase or lysine oxidase proteins in transgenic plants.

It may also be advantageous to target the tedanalactam synthase orlysine oxidase proteins to the mitochondria. This may be accomplished byfusing a suitable mitochondrial targeting peptide (MTP) to theamino-terminus of either the tedanalactam synthase or lysine oxidaseproteins. One example of an MTP that has been demonstrated to targetheterologous proteins to mitochondria of dicots is from the beta subunitof the mitochondrial ATP synthase (Boutry et al., 1987). We infer thatthis same sequence will direct mitochondrial import of either thetedanalactam synthase or lysine oxidase proteins in transgenic dicotplants when fused in frame to the amino-termini of these proteins.Appropriate promoter and termination sequences, Agrobacterium vectors,and transformation procedures needed to obtain transgenic dicotyledonousplants expressing the MTP fusion genes were described previously.

It may similarly be desirable to target the tedanalactam synthase orlysine oxidase proteins to the mitochondria of monocotyledonous plantsby making fusions of monocotyledonous plant derived MTPs to theseproteins. One example of a sequence that may direct mitochondrial importin monocots is one that is substantially homologous to the maizemitochondrial ATP synthase beta subunit (Winning et al., 1990). Anotherexample would be the MTP from the maize superoxide dismutase isozyme 3(Sod3) (White and Scandalios, 1989).

For example, the XbaI/NcoI fragment containing the portion of the maizeATP synthase beta subunit MTP sufficient to direct mitochondrial import(SEQ ID NO: 53) from pMON30447 was ligated into the XbaI and NcoI sitesof pMON25058 (lysine oxidase) that are located between the hsp70 intronand lysine oxidase coding region to obtain in-frame fusions of the maizeATP synthase beta subunit MTP (zmBATP) to the N-termini of lysineoxidase, producing plasmid pMON33700 (FIG. 26). The pMON33700 NotIcassette containing the zmBATP-lysine oxidase expression cassette wasengineered into pMON33701 (FIG. 27) to obtain pMON33702, a planttransformation vector containing the zmBATP-Lysine oxidase expressioncassette as well as previously described neomycin phosphotransferase andtedanalactam synthase expression cassettes (FIG. 28).

Transgenic corn expressing both lysine oxidase and tedanalactam synthasein roots were obtained with pMON33702. Phenotypic analysis of theselines indicated that use of the zmBATP targeting signal did notalleviate stunting associated with high level lysine oxidase expressionin leaves. However, root specific or enhanced expression of themitochondrial targeted lysine oxidase expression may alleviate stunting.It is also possible that a strategy similar to that described abovecould be used in combination with plant protease insensitive variants oflysine oxidase proenzyme to obtain transgenic maize that are notstunted.

Example 12

This example illustrates apoplastic, vacuolar, endoplastic reticulum,and peroxisomal targeting of lysine oxidase proenzyme or enzyme.

To target the lysine oxidase proprotein to the extracellular orapoplastic space, a secretory signal peptide sequence derived fromplants can be fused in frame to the amino terminus of the lysine oxidaseproprotein gene. One example of such a sequence is the signal peptidederived from a barley cysteine endoproteinase gene (Koehler and Ho,1990). Another example is the tobacco PR1b signal peptide (Cornelissenet al., 1986).

It is also recognized that both the lysine oxidase and tedanalactarnsynthase proteins contain N-linked glycosylation sites that are not usedin the native proteins derived from Trichoderma. To avoid inactivationcaused by N-glycosylation of secreted lysine oxidase or tedanalactamsynthase, the glycosylation sites of these proteins could be eliminatedby site directed mutagenesis. More specifically, all or a subset of theamino acid sequences N(×)S/T could be converted to N(×)A by replacingthe native Ser or Thr codon for an Ala codon. For the lysine oxidaseproprotein (SEQ ID NO: 46), this would entail conversion of either allor a subset of T140, T325, S373, T391, and T423 to alanine or anotherresult effective amino acid residue. Conversion of lysine oxidaseresidues N138, N323, N371, N389, and N421 to glutamine (Q) residuesresults in loss of enzymatic activity, indicating that this particularset of substitutions is not preferred. For the tedanalactam synthaseprotein (SEQ ID NO: 41), S188 and S424 could be converted to alanineresidues.

Having constructed apoplastically targeted, glycosylation deficientlysine oxidase or tedanalactam synthase proteins, it is also possible toretain the proteins in the endoplasmic reticulum. This could beaccomplished by an in frame fusion of DNA sequence encoding the peptidesequence KDEL to the C-termini of the apoplastically targeted lysineoxidase or tedanalactam synthase coding sequences (Munro and Pelham,1987; Tillmann et al., 1989). It would also similarly be possible toachieve vacuolar localization via in frame fusions of vacuolar targetingsignals (Bednarek et al., 1990; Neuhaus et al., 1991) to the C-terminiof the apoplastically targeted, glycosylation deficient lysine oxidaseor tedanalactam synthase proteins.

To target lysine oxidase enzyme or proenzyme to peroxisomes, theC-terminal peroxisomal targeting sequences could be fused in frame tothe C-terminus of lysine oxidase (Gould et al., 1987; Volokita, 1991).In this example, an amino terminal peroxisomal targeting signal (nPTS)derived from a rice malate dehydrogenase gene (Seq ID NO: 54; SEQ ID NO:55) was fused to the N-terminus of the lysine oxidase proenzyme with sixN-terminal histidine residues. A plant expression cassette consisting ofthe 4as-1 promoter, the rice actin intron, the wheat CAB leader, thenPTS-lysine oxidase fusion gene, and a wheat tahsp 17 3′ polyadenylationsite was constructed in pMON25092. The pMON25092 NotI cassettecontaining the nPTS-lysine oxidase expression cassette was engineeredinto pMON33701 to obtain pMON38800, a plant transformation vectorcontaining the nPTS-Lysine oxidase expression cassette as well aspreviously described neomycin phosphotransferase and tedanalactamsynthase expression cassettes (FIG. 29).

Transgenic corn expressing both lysine oxidase and tedanalactam synthasein roots were obtained with pMON38800. Phenotypic analysis of this linesindicated that use of the nPTS targeting signal did not alleviatestunting associated with high level lysine oxidase expression in leaves.However, root specific or enhanced expression of the mitochondrialtargeted lysine oxidase expression may alleviate stunting. It is alsopossible that a strategy similar to that described above could be usedin combination with plant protease insensitive variants of lysineoxidase proenzyme to obtain transgenic maize that are not stunted.

Example 13

This example illustrates activation of lysine oxidase proprotein by cornrootworm midgut proteases and engineering of improved proenzymevariants.

The lysine oxidase gene encodes a 69 kDa proenzyme which is N-terminallycleaved when expressed in Trichoderma or Saccharomyces to yield a matureand enzymatically active protein of approximately 60 kDa. To determineif the lysine oxidase-69 (LO69) proenzyme (Seq ID NO: 46) expressed incorn protoplasts can be activated by the proteases present in the midgutof southern corn rootworm, midguts were dissected from late instarlarvae and the proteases extracted. Protease activity assays confirmedthe presence of proteases in this extract. This extract and appropriatecontrols were incubated for about 22 hours at 25° C. with extracts fromprotoplasts electroporated with pMON25040 (i.e. the CaMV35S promoter andhsp70 intron fused to the 1.9 kb lysine oxidase gene encoding the M_(r)69,000 proprotein, SEQ ID NO:45) and non-electroporated controls andassayed for lysine oxidase activity. These results are summarized inTable 20.

TABLE 20 Lysine oxidase activity in corn leaf protoplast extractstreated with southern corn rootworm midgut extracts. Lysine oxidase FoldSample Treatment Sp. Act. Difference¹ LO69 gut extract 1292 U/μg protein130 LO69 gut extract, 997  97 protease inhibitor LO69 BSA 12 1.2 LO69heated gut extract 10 0 control all above treatments  0 NA ¹Folddifference is relative to the LO69 with heated gut extract NA = notapplicable

To confirm that the observed increase in lysine oxidase activity was dueto proteolysis of the proprotein, the LO69 and control extracts treatedwith both active and heated gut extract were analyzed by Western blotwith antibodies directed against lysine oxidase. LO69 extract treatedwith the gut extract yielded a band that migrates with the “mature”lysine oxidase of approximately M_(r) 60,000 while LO69 extracts treatedwith heated gut extract yield a much larger band of approximately M_(r)69,000. The Western data thus support our hypothesis that the CRW gutextracts activate the LO69 proenzyme by proteolysis.

These results indicate that expression of the LO69 zymogen in transgenicplants may prevent deleterious effects of lysine oxidase on plant growthand development. However, ingestion of LO69 expressing root tissue byCRW is likely to result in activation of lysine oxidase by proteolysisand subsequent insecticidal activity when the tedanalactam synthaseprotein is also present. To test the hypothesis that enzymaticallyinactive LO69 has insecticidal activity, LO69 proenzyme (LO69) waspurified from a heterologous Saccharomyces cerevisiae system and testedfor bioactivity in a diet overlay assay with SCRW (Table 21). Theabsence of LO activity in the proenzyme sample was confirmed by directenzymatic assays just prior to exposure to insects. The results of thisexperiment indicate that SCRW growth is inhibited by ingestion ofenzymatically inactive LO69 protoxin, supporting the concept thatenzymatically inactive LO69 could be produced within transgenic plantsto yield control of target insects upon ingestion.

TABLE 21 Bioactivity of the LO69 proenzyme when combined withTedanalactam Synthase (50 kD) against Southern Corn Rootworm Larvae Av.Wght % Sample (ppm)¹ n Surv (mg) SEM² inhibition³ Tris control 18 18 2.60.48 50 kD (2) 18 18 3.5 0.46 0 50 kD (10)** 18 18 1.5 0.39 39 LO69 (2)18 17 2.1 0.56 20 LO69 (10) 17 17 3.3 0.52 0 LO69 (20) 18 18 2.8 0.51 050 kD (2)/LO (2) 18 18 1.6 0.37 39 50 kD (2)/LO69 (2) 18 18 1.5 0.4 4150 kD (2)/LO69 (10) 18 18 0.34 0.03 87 50 kD (2)/LO69 (20) 18 15 0.590.26 77 ¹Sample Key: 50 kD = tedanalactam synthase; LO69 = LO proenzyme;LO = LO (active enzyme); (2) = concentration of 2 ppm ²SEM is standarderror of measurement ³% inhibition is: Control Larval Weight − TestLarval Weight/Control Larval Weight × 100. **Low level LO contaminationin the Trichoderma derived 50 kD sample accounts for the bioactivityobserved at this concentration of the 50 kD preparation.

Note that the stability of the native LO69 zymogen in planta may beimproved by changing the amino acid sequences in the proprotein regionthat are recognized by plant proteases. These sequences can beidentified by obtaining the N-terminal protein sequence of enzymaticallyactive lysine oxidase (i.e. the approximately M_(r) 60,000 proteinproduced by proteolysis of the M_(r) 69,000 proprotein) isolated fromtransgenic plants. For example, mature and enzymatically active lysineoxidase from roots of corn transformed with pMON25040 or pMON30417 isapparently produced by cleavage at the C-terminus of the glu 80 residue.A mutagenized or improved LO69 proenzyme would display greater stabilityin transgenic plants, resulting in lower levels of enzymatically activelysine oxidase and decreased impact of plant growth and/or lysinecontent. The improved LO69 proenzyme would retain amino acid sequencesrecognized by coleopteran midgut proteases, resulting in full activationand biological activity upon ingestion (Table 22).

TABLE 22 Design of lysine oxidase proenzyme variants resistant to plantproteases yet susceptible to target insect midgut proteases NameSequence Type SEQ ID NO WT1 N-KPALLKEAPRAEEELPPRK-C wild type 46 mut1N-KPALLGGGGXXXXXXGGGG-C mutant 56 mut2 N-GGGSGGXXXXXXGGGPPRK-C mutant 57mut3 N-KPGGGGXXXXXXGGGPPRK-C mutant 58 (X is any of the 20 naturallyoccurring amino acid residues)

It is also recognized that amino acid residues lys 68 through lys 86 ofthe lysine oxidase proenzyme (SEQ ID NO: 46) represent a proteasesensitive region in the lysine oxidase proprotein or zymogen. Variantsin this region may yield the desired characteristic of being susceptibleto CRW gut proteases, yet resistant to plant root proteases. Onepreferred embodiment of this invention would be the substitution of thisentire region of nineteen (19) amino acids with a peptide sequence thatrepresents an optimal corn rootworm gut protease cleavage site.Potential representation of this type of amino acid substitutions areshown in SEQ ID. NOS: 56-58. Methods for identifying optimal proteasesubstrates in combinatorial peptide libraries have been identified andcould be employed (Duan and Laursen, 1994). For example, a library ofprotease substrates consisting of a randomized target protease cleavagesite separating a floresence donor and acceptor pair can be immobilizedon a cellulose filter. This filter is then exposed to plant and insectgut proteases; peptides that floresce only in the presence of insectproteases would represent preferred substrates.

It is finally recognized that the entire lysine oxidase proenzymesequence (SEQ ID NO: 46) extending from amino acid residues 1 (met) to87 (val) may be modified to encode a variant with the desired propertyof being resistant to activation by corn root proteases yet sensitive toactivation by corn rootworm gut proteases. This region could bemutagenized by either site-directed or random mutagenesis techniquesfamiliar to those skilled in the art (Kunkel, 1985; Spee et al, 1993;Muhlrad et al, 1992). The population of mutagenized lysine oxidaseproprotein expressing clones expressed in Saccharomyces cerevisiae couldpotentially be screened via exposure to plant and insect proteasesfollowed by lysine oxidase enzymatic assays to identify the variant withthe desired protease activation properties. Finally, the improved lysineoxidase zymogen could be targeted to plastids, mitochondria, theapoplastic space, or the vacuole as described above.

The lysine oxidase proenzyme sequence extending from amino acids 1 to 87(SEQ ID NO: 46) may also be fused to proteins other than lysine oxidaseto create other zymogens that would be activated upon insect ingestion.The resultant chimeric protein could then be activated by coleopteran orlepidopteran midgut proteases to yield enzymatically active,insecticidal proteins.

All of the compositions and methods disclosed and claimed herein can bemade and executed without undue experimentation in light of the presentdisclosure. While the compositions and methods of this invention havebeen described in terms of preferred embodiments, it will be apparent tothose of skill in the art that variations may be applied to thecompositions and methods and in the steps or in the sequence of steps ofthe methods described herein without departing from the concept, spiritand scope of the invention. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the spirit, scope and concept of the invention as defined by theappended claims.

In view of the above, it will be seen that the several advantages of theinvention are achieved and other advantageous results attained.

As various changes could be made in the above methods and compositionswithout departing from the scope of the invention, it is intended thatall matter contained in the above description and shown in theaccompanying drawings shall be interpreted as illustrative and not in alimiting sense.

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The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

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58 22 amino acids amino acid Not Relevant linear 1 Asp Ala Pro Pro GlnPro Pro Lys Glu Asp Glu Leu Val Glu Leu Ile 1 5 10 15 Leu Gln Asn LeuAla Arg 20 20 base pairs nucleic acid single linear modified_base 12/mod_base= i modified_base 15 /mod_base= i 2 TCRTCYTCYT TNGGNGGYTG 20 13amino acids amino acid Not Relevant linear 3 Gly Leu Asn Leu His Pro ThrGln Ala Asp Ala Ile Arg 1 5 10 23 base pairs nucleic acid single linearmodified_base /mod_base= i modified_base /mod_base= i modified_base 15/mod_base= i modified_base 18 /mod_base= i 4 ATNGCRTCNG CYTGNGTNGG RTG23 23 base pairs nucleic acid single linear modified_base /mod_base= imodified_base /mod_base= i modified_base 15 /mod_base= i modified_base21 /mod_base= i 5 CAYCCNACNC ARGCNGAYGC NAT 23 20 base pairs nucleicacid single linear modified_base /mod_base= i modified_base 12/mod_base= i modified_base 15 /mod_base= i 6 AAYCTNCAYC CNACNCARGC 20 20base pairs nucleic acid single linear modified_base 12 /mod_base= imodified_base 15 /mod_base= i 7 AAYTTRCAYC CNACNCARGC 20 9 amino acidsamino acid Not Relevant linear 8 Lys Gln Gln Ala Phe Gly Tyr Tyr Lys 1 520 base pairs nucleic acid single linear modified_base 12 /mod_base= imodified_base 18 /mod_base= i 9 AARCARCARG CNTTYGGNTA 20 20 base pairsnucleic acid single linear modified_base /mod_base= i modified_base 12/mod_base= i 10 CARGCNTTYG GNTAYTAYAA 20 12 amino acids amino acid NotRelevant linear 11 Tyr Pro Ser Tyr Asn Xaa Asp Asp Thr Gly Glu Ala 1 510 763 base pairs nucleic acid single linear 12 AAGCAGCAGG CGTGGGTATTACAAAGAGAA GCTTGCTGAG GACTTCGACA AAGGGTTCGA 60 TGAGCTCATG CTCGTCGACGACATGACCAC TCGAGAGTAC TTGAAGCGAG GCGGGCCGAA 120 GGGAGAGGCG CCCAAGTATGACTTTTTCGC CATCCAGTGG ATGGAGACAC AAAACACTGG 180 GACAAACCTG TTTGATCAGGCCTTTTCTGA AAGCGTCATC GACTCGTTTG ACTTTGACAA 240 CCCGACAAAG CCCGAATGGTACTGCATCGA GGGAGGAACA TCGCTTTTGG TGGACGCCAT 300 GAAAGAAACC CTTGTCCACAAGGTACAGAA CAACAAGAGA GTTGATGCCA TTTCCATTGA 360 CTTGGACGCT CCGGATGATGGGAACATGTC GGTCAGGATA GGCGGAAAGG ATCACTCCGG 420 ATATAGCACC GTCTTCAACACCACCGCTCT GGGCTGCCTT GACCGCATGG ATCTGCGTGT 480 CTCAACTTGC ACCCTACTCAGGCAGATGCC ATTCGATGTT TGCACTATGA CAACTCGACC 540 AAGGTGGCTC TCAAGTTTACTACCCGTGGT GGATCAAGGA CTGTGGCATC ACTTGCGGTG 600 GCGCGGCCTC GACTGATCTACCTCTACGAA CTTGCGTTTA CCCATCATAC AACTTGGACG 660 ATACTGGTGA GGCTGTTCTGCTTGCCTCAT ACACTTGGTC TCAAGATGCA ACTCGCATTG 720 GATCGTTGGT GAAGGACGCTCCACCACACC CCCCAAAGAA GAC 763 23 base pairs nucleic acid single linear13 ACCTCTACGA ACTTGCGTTT ACC 23 23 base pairs nucleic acid single linear14 CAACTCGCAT TGGATCGTTG GTG 23 2700 base pairs nucleic acid singlelinear 15 GATCTAACCA CGGCTTTTGC GCTCCAGGCC GCTCCCACCG CATGCAGGCATTCCATCCCA 60 ACCCTCAAAT GAGTCTAGCC TTCAGCCTTC ACCTGCAAGT GGCTGGCGGGATGTGTTCGG 120 GACTTCATGC AGCTCAAGAC TCTGAGCCTC GCTGATGATG AGGGGATTCAAGACATGCAT 180 TTCAGCTTTG GTGATAAGAG CCAATAGTGT TTGCTGCTCA TGTTGTCTGTGCTTTCTGTG 240 CCGCTTCTGT GCCGTTATCG CCTGTTTTAT AGCGTCAGCC AAGCCAATCAGTCTCCTCCC 300 GCTGGAATCC CTCCCGTGTC ATTTTTCTCC CCGTTACGCA ATTCTTCCTTAATCGATACT 360 ACTATACAGT ATGATGGAGA GCTTTTACTG GTGCCCACTT TGTGGCAATGCTATTGATGT 420 CTTTCAAGTC AGAGCTGAGC ACGGAAATCG ATAGCCTGAC CTCTAACGGCTGTCGGTAGC 480 TGAAAGGGGA TGAGAGCGGA GGCGGTTAAT TCAGCTAGGT ATTGATTAAGGGAACTGGCA 540 GCTTGTGTTC ACGTAGGCTC TGAATAAGAT ATAAATAAGG AGAGGAAAGGCTACGCAATC 600 GAAGTAAACG GCTACCATCG CCATCTTCTC ATCATAGCTA TCCCGTTACTATATTTGCAA 660 ACATGGACAA TGTTGACTTT GCTGAATCTG TCCGAACCCG CTGGGCGAGGCGACTCATTC 720 GTGAGAAGGT CGCCAAGGAA CTCAACATTC TAACCGAAAG ACTTGGTGAGGTGCCCGGCA 780 TCCCTCCTCC AAATGAAGGC AGGTTCCTGG GCGGCGGCTA CTCTCACGACAATCTACCGT 840 CTGATCCCCT CTATTCCAGC ATTAAGCCGG CTCTTCTAAA GGAGGCTCCTCGAGCAGAAG 900 AGGAACTGCC GCCTCGAAAG GTGTGCATCG TAGGCGCTGG TGTTTCCGGCCTCTACATAG 960 CCATGATTTT GGATGATTTG AAAATCCCAA ATCTCACTTA CGACATCTTCGAATCCAGTT 1020 CCAGAACTGG TGGCCGTTTG TATACGCACC ATTTCACCGA CGCCAAGCATGACTATTACG 1080 ACATTGGTGC TATGCGATAT CCTGACATCC CCAGCATGAA ACGTACCTTTAACCTGTTTA 1140 AACGTACTGG GATGCCTCTC ATCAAATATT ACCTTGATGG CGAGAATACCCCTCAGCTGT 1200 ACAATAATCA CTTCTTCGCC AAGGGCGTGT CGGACCCCTA TATGGTGAGCGTGGCCAATG 1260 GCGGCACCGT GCCAGATGAT GTTGTCGATA GTGTTGGAGA GAAGTTACAACAGGCTTTCG 1320 GTTATTACAA AGAGAAGCTT GCTGAGGACT TCGACAAAGG GTTCGATGAGCTCATGCTCG 1380 TCGACGACAT GACCACTCGA GAGTACTTGA AGCGAGGCGG GCCGAAGGGAGAGGCGCCCA 1440 AGTATGACTT TTTCGCCATC CAGTGGATGG AGACACAAAA CACTGGGACAAACCTGTTTG 1500 ATCAGGCCTT TTCTGAAAGC GTCATCGACT CGTTTGACTT TGACAACCCGACAAAGCCCG 1560 AATGGTACTG CATCGAGGGA GGAACATCGC TTTTGGTGGA CGCCATGAAAGAAACCCTTG 1620 TCCACAAGGT ACAGAACAAC AAGAGAGTTG ATGCCATTTC CATTGACTTGGACGCTCCGG 1680 ATGATGGGAA CATGTCGGTC AGGATAGGCG GAAAGGATCA CTCCGGATATAGCACCGTCT 1740 TCAACACCAC CGCTCTGGGC TGCCTTGACC GCATGGATCT GCGTGGTCTCAACTTGCACC 1800 CTACTCAGGC AGATGCCATT CGATGTTTGC ACTATGACAA CTCGACCAAGGTGGCTCTCA 1860 AGTTTAGCTA CCCGTGGTGG ATCAAGGACT GTGGCATCAC TTGCGGTGGCGCGGCCTCGA 1920 CTGATCTACC TCTACGAACT TGCGTTTACC CATCATACAA CTTGGACGATACTGGTGAGG 1980 CTGTTCTGCT TGCCTCATAC ACTTGGTCTC AAGATGCAAC TCGCATTGGATCGTTGGTGA 2040 AGGACGCTCC ACCACAGCCG CCCAAGGAGG ATGAGCTTGT CGAGCTGATCCTGCAGAACC 2100 TAGCCCGCCT GCACGCTGAG CATATGACCT ACGAGAAGAT TAAGGAGGCTTACACGGGCG 2160 TATATCACGC CTATTGCTGG GCTAATGATC CCAATGTCGG TGGTGCTTTCGCCCTCTTCG 2220 GTCCCGGCCA GTTCAGCAAT CTGTATCCAT ACCTGATGCG GCCAGCGGCGGGCGGCAAGT 2280 TCCATATCGT CGGAGAGGCA TCTAGTGTGC ATCACGCCTG GATCATAGGGTCTTTGGAGA 2340 GCGCTTACAC CGCTGTGTAC CAGTTCTTGT ACAAGTACAA GATGTGGGATTACTTGAGGT 2400 TGTTGTTGGA GCGCTGGCAG TATGGTCTCC AGGAGTTAGA GACGGGGAAGCACGGTACGG 2460 CTCATTTGCA GTTTATTCTA GGTTCACTTC CCAAGGAGTA CCAGGTGAAGATTTAAAGCG 2520 AAAGAGGTAC TACGGCATGG AGACAATTTT GGGTAGAGAT TCTAGTATTCCAGCAGTTTC 2580 ATAGAAAGTG TGATGTTTGT TAGTCCCACT TTGAGTCTCT GTTCGTCTGAAAGTGCCTAC 2640 TATGACCCGG TGATTAGTAT AACAGAATTT GTCATTCTCA TCAGCCATAAACCGAGGTCA 2700 22 base pairs nucleic acid single linear 16 CATGTCGTCGACGAGCATGA GC 22 23 base pairs nucleic acid single linear 17 CATCGAACCCTTTGTCGAAG TCC 23 25 base pairs nucleic acid single linear 18 CAGCAAGCTTCTCTTTGTAA TACCC 25 274 base pairs nucleic acid single linear 19ACTATACGAC ATTGGTGCTA TGCGATATCC TGACATCCCC AGCATGAAAC GTACCTTTAA 60CCTGTTTAAA CGTACTGGGA TGCCTCTCAT CAAATATTAC CTTGATGGCG AGAATACCCC 120TCAGCTGTAC AATAATCACT TCTTCGCCAA GGGCGTGTCG GACCCCTATA TGGTGAGCGT 180GGCCAATGGC GGCACCGTSC CAGATGATGT TNGTCGATAG TGTTGGAGAG AAGTTACAAC 240AGGCTTTCGG GTATTACAAA GAGAAGCTTG CTGA 274 31 base pairs nucleic acidsingle linear 20 GTCGAAGTCC TCAGCCAGCT TCTCTTTGTA A 31 18 base pairsnucleic acid single linear 21 CATGCTGGGG ATGTCAGG 18 262 base pairsnucleic acid single linear 22 ACCATCGCCA TCTTCTCATC ATAGCTATCCCGTTACTATA TCTGCAAACA TGGACAATGT 60 TGACTTTGCT GAATCTGTCC GAACCCGCTGGGCGAGGCGA CTCATTCGTG AGAAGGTCGC 120 CAAGGAACTC AACATTCTAA CCGAAAGACTTGGTGAGGTG CCCGGCATCC CTCCTCCAAA 180 TGAAGGCAGG TTCCTGGGCG GCGGCTACTCTCTCGACAAT CTACCGCCTG ATCCCCTCTA 240 TTCCAGCATT AAGCCGGCTC TT 262 26base pairs nucleic acid single linear 23 GARCARAAYA AYTTYTTYAA YCAYGC 2620 amino acids amino acid Not Relevant linear 24 Val Val Val Leu Glu GlnAsn Asn Phe Phe Asn His Ala Gly Ser Ser 1 5 10 15 Asn Asp Leu Ala 20 21base pairs nucleic acid single linear modified_base /mod_base= i 25ATGTAYACNG ARCAYTAYAT G 21 13 amino acids amino acid Not Relevant linear26 Thr Met Tyr Thr Glu Asp Tyr Met Ala Asp Leu Ala Lys 1 5 10 20 basepairs nucleic acid single linear modified_base /mod_base= i 27GGNGCRAAYT GRAACCACAT 20 15 amino acids amino acid Not Relevant linear28 Gly Thr Ile Phe Pro Ser Met Trp Phe Gln Phe Ala Pro Asp Lys 1 5 10 15623 base pairs nucleic acid single linear 29 ATGTATACGG AGGACTACATGGCCGATCTT GCCAAGGAAG CCTTGGCCCT CTGGGATGAT 60 CTTGAGAGAG ATTCCGGTACGCCACTGCGA TGGATGAGCG GCCTCCTCAA CTTTGGCGAT 120 AAGGACTATG GCGGCGATACACCCGAAGGA ACCTTGTTGG GGCCAATTGC GAACCTGGAC 180 CGCCTGGGAA TGACTTATCAAGAGTTATCT GCTAAGGAGA TTGAGGCACG CTACCCGTTC 240 AAGAACCTCG ACCCTAAGTACATTGGTCTC TTCGCGCCAG ACAATGGCGT CATCAATGTC 300 CAGCTTCTGT TGAGGACGCTGTATAAATTA TCACTGGACT ATGGTGCCAC TGCGAAACAG 360 CATACCAAAG TCCAGGCTATTAAGCCTTCT AATCATTCTC ATTACGCCTG GGATGTTCAC 420 GCTATTCGTC ATGAGACCGAAGCCGCTGTC TTCAAGGCAA AGAAGATCAT TATCGCCTCT 480 GGTGCTTACG TGAACCATGTTCTCAAGCCG AGCTTCGACA TTTCTCTCGA TCTCGACATC 540 TGAGAAATGG TGTTTTCTTACTTTAACTGC AATGCAGGAC CCAAAGGAAC AATATTCCCC 600 AGCATGTGGT TCCAATTCGCCCC 623 11 amino acids amino acid Not Relevant linear 30 Leu Gly Met ThrTyr Gln Glu Met Ser Ala Lys 1 5 10 27 base pairs nucleic acid singlelinear 31 CCGGAATTCC TTGGCAAGAT CGGCCAT 27 19 base pairs nucleic acidsingle linear 32 CCTCCGTATA CATTGTTCG 19 381 base pairs nucleic acidsingle linear 33 GAATTCGGCT TCTACTACTA CTAGGCCACG CGTCGACTAG TACGGGGGGGGGGGGGGTGG 60 GGGTGACATC ACGTTGTTTC AGTGCTGGAT ATAGGTTCCT CCTAGAGTTTACCTATTGAG 120 ACAGATACTT CAATCACATT CTCTAGGATA TCGAATCAAA CCGAAAACACTTGCTTCAGA 180 ATCCCCTAAA CATGGCAGAC GAAATCTACG ATGTTGTCGT CATCGGCGGCGGCCCAATTG 240 GATTGGCAGC TGCCTATGAA GCAGCCAAGG AGGGTGCCAA AGTCGTTGTTCTCGAGCAAA 300 ACAATTTCTT CAACCATGCT GGGAGCTCTA ACGATTTGGC TCGGATGTTTCGAACAATGT 360 ATACGGAGGA AGCCGAATTC C 381 31 base pairs nucleic acidsingle linear 34 CCGGAATTCA TGGCCGATCT TGCCAAGGAA G 31 1426 base pairsnucleic acid single linear 35 GAATTCGGCT TCCGGAATTC ATGGCCGATCTTGCCAAGGA AGCCTTGGCC CTCTGGGATG 60 ATCTTGAGAG AGATTCCGGT ACGCCACTGCGATGGATGAG CGGCCTCCTC AACTTTGGCG 120 ATAAGGACTA TGGCGGCGAT ACACCCGAAGGAACCTTGTT GGGGCCAATT GCGAACCTGG 180 ACCGCCTGGG AATGACTTAT CAAGAGTTATCTGCTAAGGA GATTGAGGCA CGCTACCCGT 240 TCAAGAACCT CGACCCTAAG TACATCGGTCTCTTCGCGCC AGACAATGGG CTCATCAATG 300 TCCAGCTTCT GTTGAGGACG CTGTATAAATTATCACTGGA CTATGGTGCC ACTGCGAAAC 360 AGCATACCAA AGTCCAGGCT ATTAAGCCTTCTAATCATTC TCATTACGCC TGGGATGTTC 420 ACGCTATTCG TCATGAGACC GAAGCCGCTGTCTTCAAGGC AAAGAAGATC ATTATCGCCT 480 CTGGTGCTTA CGTGAACCAT GTTCTCAAGCCGAGCTTCGA CATTTCTCTC GATCTCGACA 540 TCTGGGAAAT GGTGTTTTCT TACTTTAACTGCAATGCAGG ACCCAAAGGA ACAATATTCC 600 CCAGCATGTG GTTCCAGTTT GCGCCTGATAAGAACGGCAG ATCACAGCTC TTCTATGGCT 660 TTCCAGCACT TCCATGGGGC CCTCCAAATCTTGCTCGTAT TGCTATGGAT GCGGCCACCA 720 GGCGGATCAA GGATCCCAAC GAGAGACTTACAAGCACTAT TAACCCGGAG GATATTGCTG 780 ATACGCAAGA GTTTATCCGC AATCATTGTGTCAACGTTGA TCCTACCATT CCTGCGTTGA 840 CATCGAGTTG CCTGCAGACC AATGTGTTTGACAACATGTT TGTTCTGGAC TTTGTCCCTG 900 AAAAATATCT GAACGGCGGA GCCAAAGACAGTGTAGTCGT CTTCACAGCC GGATGGGCCA 960 TGAAGTTCGT GCCAATGATA GGAAAGGCACTCGCTGACAT GGCACTCAAG GGAAGCTCTC 1020 CATATGCGCG CAAAGAATTT GCCATCACCCGCACAGATTC AGCGACCGGG AAGGGCATCA 1080 TTGTGGAAGG TGGATCAGAG AACCGATCGGTTAAGAGCAG CGCTTTTGTC TTCTACTCAC 1140 CAGGCATCCG GTTCTTCGTT TGCCGGCTTCCATAACACTG CACGGCAATA GAAGAAAGTG 1200 AATAGGGGTA AGCGGGCGGG ATAGGATATCTGTGGAACAC ACAATGAGAA GTGACCAAGA 1260 TCGCTGTTGA GAATACGCCA AAGCATACTATAGCTTGTAG GTGTTGCTAT CTGGTCTACA 1320 GTGTTGCAAA GATGCATAAA TAGGTGAAAAAGAATTGATG AGGTATATGA ATCCTCAGTA 1380 AAAAAAAAAA AAAAAAATCG ATGTCGACTCGAGTCAAGCC GAATTC 1426 27 base pairs nucleic acid single linear 36GGGAGATCTC CATGGCAGAC GAAATCT 27 31 base pairs nucleic acid singlelinear 37 GGCTTTCCAG CACTTCCTTG GGGCCCTCCA A 31 31 base pairs nucleicacid single linear 38 TTGGAGGGCC CCAAGGAAGT GCTGGAAAGC C 31 31 basepairs nucleic acid single linear 39 CCCAAGCTTG AATTCACTTT CTTCTATTGC C31 1385 base pairs nucleic acid single linear 40 GGGAGATCTC CATGGCAGACGAAATCTACG ATGTTGTCGT CATCGGCGGC GGCCCAATTG 60 GATTGGCAGC TGCCTATGAAGCAGCCAAGG AGGGTGCCAA AGTCGTTGTT CTCGAGCAAA 120 ACAATTTCTT CAACCATGCTGGGAGCTCTA ACGATTTGGC TCGGATGTTT CGAACAATGT 180 ATACGGAGGA TTATATGGCCGATCTTGCCA AGGAAGCCTT GGCCCTCTGG GATGATCTTG 240 AGAGAGATTC CGGTACGCCACTGCGATGGA TGAGCGGCCT CCTCAACTTT GGCGATAAGG 300 ACTATGGCGG CGATACACCCGAAGGAACCT TGTTGGGGCC AATTGCGAAC CTGGACCGCC 360 TGGGAATGAC TTATCAAGAGTTATCTGCTA AGGAGATTGA GGCACGCTAC CCGTTCAAGA 420 ACCTCGACCC TAAGTACATTGGTCTCTTCG CGCCAGACAA TGGGCTCATC AATGTCCAGC 480 TTCTGTTGAG GACGCTGTATAAATTATCAC TGGACTATGG TGCCACTGCG AAACAGCATA 540 CCAAAGTCCA GGCTATTAAGCCTTCTAATC ATTCTCATTA CGCCTGGGAT GTTCACGCTA 600 TTCGTCATGA GACCGAAGCCGCTGTCTTCA AGGCAAAGAA GATCATTATC GCCTCTGGTG 660 CTTACGTGAA CCATGTTCTCAAGCCGAGCT TCGACATTTC TCTCGATCTC GACATCTGGG 720 AAATGGTGTT TTCTTACTTTAACTGCAATG CAGGACCCAA AGGAACAATA TTCCCCAGCA 780 TGTGGTTCCA GTTTGCGCCTGATAAGAACG GCAGATCACA GCTCTTCTAT GGCTTTCCAG 840 CACTTCCTTG GGGCCCTCCAAATCTTGCTC GTATTGCTGT GGATGCGGCC ACCAGGCGGA 900 TCAAGGATCC CAACGAGAGACTTACAAGCA CTATTAACCC GGAGGATATT GCTGATACGC 960 AAGAGTTTAT CCGCAATCATTGTGTCAACG TTGATCCTAC CATTCCTGCG TTGACATCGA 1020 GTTGCCTGCA GACCAATGTGTTTGACAACA TGTTTGTTCT GGACTTTGTC CCTGAAAAAT 1080 ATCTGAACGG CGGAGCCAAAGACAGTGTAG TCGTCTTCAC AGCCGGATGG GCCATGAAGT 1140 TCGTGCCAAT GATAGGAAAGGCACTCGCTG ACATGGCACT CAAGGGAAGC TCTCCATATG 1200 CGCGCAAAGA ATTTGCCATCACCCGCACAG ATTCAGCGAC CGGGAAGGGC ATCATTGTGG 1260 AAGGTGGATC AGAGAACCGATCGGTTAAGA GCAGCGCTTT TGTCTTCTAC TCACCAGGCA 1320 TCCGGTTCTT CGTTTGCCGGCTTCCATAAC ACTGCACGGC AATAGAAGAA AGTGAATTCA 1380 AGCTT 1385 445 aminoacids amino acid Not Relevant linear 41 Met Ala Asp Glu Ile Tyr Asp ValVal Val Ile Gly Gly Gly Pro Ile 1 5 10 15 Gly Leu Ala Ala Ala Tyr GluAla Ala Lys Glu Gly Ala Lys Val Val 20 25 30 Val Leu Glu Gln Asn Asn PhePhe Asn His Ala Gly Ser Ser Asn Asp 35 40 45 Leu Ala Arg Met Phe Arg ThrMet Tyr Thr Glu Asp Tyr Met Ala Asp 50 55 60 Leu Ala Lys Glu Ala Leu AlaLeu Trp Asp Asp Leu Glu Arg Asp Ser 65 70 75 80 Gly Thr Pro Leu Arg TrpMet Ser Gly Leu Leu Asn Phe Gly Asp Lys 85 90 95 Asp Tyr Gly Gly Asp ThrPro Glu Gly Thr Leu Leu Gly Pro Ile Ala 100 105 110 Asn Leu Asp Arg LeuGly Met Thr Tyr Gln Glu Leu Ser Ala Lys Glu 115 120 125 Ile Glu Ala ArgTyr Pro Phe Lys Asn Leu Asp Pro Lys Tyr Ile Gly 130 135 140 Leu Phe AlaPro Asp Asn Gly Leu Ile Asn Val Gln Leu Leu Leu Arg 145 150 155 160 ThrLeu Tyr Lys Leu Ser Leu Asp Tyr Gly Ala Thr Ala Lys Gln His 165 170 175Thr Lys Val Gln Ala Ile Lys Pro Ser Asn His Ser His Tyr Ala Trp 180 185190 Asp Val His Ala Ile Arg His Glu Thr Glu Ala Ala Val Phe Lys Ala 195200 205 Lys Lys Ile Ile Ile Ala Ser Gly Ala Tyr Val Asn His Val Leu Lys210 215 220 Pro Ser Phe Asp Ile Ser Leu Asp Leu Asp Ile Trp Glu Met ValPhe 225 230 235 240 Ser Tyr Phe Asn Cys Asn Ala Gly Pro Lys Gly Thr IlePhe Pro Ser 245 250 255 Met Trp Phe Gln Phe Ala Pro Asp Lys Asn Gly ArgSer Gln Leu Phe 260 265 270 Tyr Gly Phe Pro Ala Leu Pro Trp Gly Pro ProAsn Leu Ala Arg Ile 275 280 285 Ala Val Asp Ala Ala Thr Arg Arg Ile LysAsp Pro Asn Glu Arg Leu 290 295 300 Thr Ser Thr Ile Asn Pro Glu Asp IleAla Asp Thr Gln Glu Phe Ile 305 310 315 320 Arg Asn His Cys Val Asn ValAsp Pro Thr Ile Pro Ala Leu Thr Ser 325 330 335 Ser Cys Leu Gln Thr AsnVal Phe Asp Asn Met Phe Val Leu Asp Phe 340 345 350 Val Pro Glu Lys TyrLeu Asn Gly Gly Ala Lys Asp Ser Val Val Val 355 360 365 Phe Thr Ala GlyTrp Ala Met Lys Phe Val Pro Met Ile Gly Lys Ala 370 375 380 Leu Ala AspMet Ala Leu Lys Gly Ser Ser Pro Tyr Ala Arg Lys Glu 385 390 395 400 PheAla Ile Thr Arg Thr Asp Ser Ala Thr Gly Lys Gly Ile Ile Val 405 410 415Glu Gly Gly Ser Glu Asn Arg Ser Val Lys Ser Ser Ala Phe Val Phe 420 425430 Tyr Ser Pro Gly Ile Arg Phe Phe Val Cys Arg Leu Pro 435 440 445 2093base pairs nucleic acid single linear 42 ATATGTCGCA TTCTGGACATTCTACGGTAT CATTATTTGT GGCGCAGTGG TTTATACGAC 60 TCAATTGAGT ATTATATTAAGCCGACATTC CGAAGGTCTT CTCTATCGCC ACATCACGTT 120 GTTTCAGTGC TGGATATAGGTTCCTCCTAG AGTTTACCTA TTGAGACAGA TACTTCAATC 180 ACATTCTCTA GGATATCGAATCAAACCGAA AACACTTGCT TCAGAATCCC CTAAACATGG 240 CAGACGAAAT CTACGATGTTGTCGTCATCG GCGGCGGCCC AATTGGATTG GCAGCTGCCT 300 ATGAAGCAGC CAAGGAGGGTGCCAAAGTCG TTGTTCTCGA GCAAAACAAT TTCTTCAACC 360 ATGCTGGGAG CTCTAACGATTTGGCTCGGA TGTTTCGAAC AATGTGAGTT ATTTTTTTGT 420 CTTTTTTCTT ACTCTCGTTTTCACAGACAC AGCTAATCAT CCGATCAGGT ATACGGAGGA 480 TTATATGGCC GATCTTGCCAAGGAAGCCTT GGCCCTCTGG GATGATCTTG AGAGAGATTC 540 CGGTACGCCA CTGCGATGGATGAGCGGCCT CCTCAACTTT GGCGATAAGG ACTATGGCGG 600 CGATACACCC GAAGGTATGAAATCCTCCCA CAATAATATG GGTTTTGGCG CCCTTGTCTC 660 ACGATTTCAA CAGGAACCTTGTTGGGGCCA ATTGCGAACC TGGACCGCCT GGGAATGACT 720 TATCAAGAGT GTAAGTTGTGGCATGTATGC GAACGACGGT ATGCCCTCGA GTGCTAATCC 780 ATCGTCTCAC AGTATCTGCTAAGGAGATTG AGGCACGCTA CCCGTTCAAG AACCTCGACC 840 CTAAGTACAT TGGTCTCTTCGCGCCAGACA ATGGGCTCAT CAATGTCCAG CTTCTGTTGA 900 GGACGCTGTA TAAATTATCACTGGACTATG GTGCCACTGC GAAACAGCAT ACCAAAGTCC 960 AGGCTATTAA GCCTTCTAATCATTCTCATT ACGCCTGGGA TGTTCACGCT ATTCGTCATG 1020 AGACCGAAGC CGCTGTCTTCAAGGCAAAGA AGATCATTAT CGCCTCTGGT GCTTACGTGA 1080 ACCATGTTCT CAAGCCGAGCTTCGACATTT CTCTCGATCT CGACATCTGG GAAATGGTGT 1140 TTTCTTACTT TAACTGCAATGCAGGACCCA AAGGAACAAT ATTCCCCAGT ACGTGGATTG 1200 ATCCATTTCT CTCGTGAGTTGGAGGTGTAT GAGCTAACTC CCATCAACTA GGCATGTGGT 1260 TCCAGTTTGC GCCTGATAAGAACGGCAGAT CACAGCTCTT CTATGGCTTT CCAGCACTTC 1320 CATGGGGCCC TCCAAATCTTGCTCGTATTG CTGTGGATGC GGCCACCAGG CGGATCAAGG 1380 ATCCCAACGA GAGACTTACAAGCACTATTA ACCCGGAGGA TATTGCTGAT ACGCAAGAGT 1440 TTATCCGCAA TCATTGTGTCAACGTTGATC CTACCATTCC TGCGTTGACA TCGAGTTGCC 1500 TGCAGACCAA TGTGTTTGGTGCGTATATTC ATATGGATGG ATTGACAAGG AAACTTACTG 1560 ATTCGGCTTA TAGACAACATGTTTGTTCTG GACTTTGTCC CTGAAAAATA TCTGAACGGC 1620 GGAGCCAAAG ACAGTGTAGTCGTCTTCACA GCCGGATGGG CCATGAAGTT CGTGCCAATG 1680 ATAGGAAAGG CACTCGCTGACATGGCACTC AAGGGAAGCT CTCCATATGC GCGCAAAGAA 1740 TTTGCCATCA CCCGCACAGATTCAGCGACC GGGAAGGGCA TCATTGTGGA AGGTGGATCA 1800 GAGAACCGAT CGGTTAAGAGCAGCGCTTTT GTCTTCTACT CACCAGGCAT CCGGTTCTTC 1860 GTTTGCCGGC TTCCATAACACTGCACGGCA ATAGAAGAAA GTGAATAGGG GGTAAGCAGG 1920 CGGGATAGGA TATCTGTGGAACACACAATG AGAAGTGACC AAGATCGCTG TTGAGAATAC 1980 GCAAAGCATA CTATAGCTTGTAGGTGTTGC TATCTGGTCT ACAGTGTTGC AAAGATGCAT 2040 AAATAGGTGA AAAAGAATTGATGAGGTATA TGAATCCTCA GTAATCTTGA GCC 2093 35 base pairs nucleic acidsingle linear 43 TTGCAAACCA TGGACAATGT TGACTTTGCT GAATC 35 33 base pairsnucleic acid single linear 44 GCCGTAGTAC CGAATTCTTA TTAAATCTTC ACC 331883 base pairs nucleic acid single linear 45 TTGCAAACCA TGGACAATGTTGACTTTGCT GAATCTGTCC GAACCCGCTG GGCGAGGCGA 60 CTCATTCGTG AGAAGGTCGCCAAGGAACTC AACATTCTAA CCGAAAGACT TGGTGAGGTG 120 CCCGGCATCC CTCCTCCAAATGAAGGCAGG TTCCTGGGCG GCGGCTACTC TCACGACAAT 180 CTACCGTCTG ATCCCCTCTATTCCAGCATT AAGCCGGCTC TTCTAAAGGA GGCTCCTCGA 240 GCAGAAGAGG AACTGCCGCCTCGAAAGGTG TGCATCGTAG GCGCTGGTGT TTCCGGCCTC 300 TACATAGCCA TGATTTTGGATGATTTGAAA ATCCCAAATC TCACTTACGA CATCTTCGAA 360 TCCAGTTCCA GAACTGGTGGCCGTTTGTAT ACGCACCATT TCACCGACGC CAAGCATGAC 420 TATTACGACA TTGGTGCTATGCGATATCCT GACATCCCCA GCATGAAACG TACCTTTAAC 480 CTGTTTAAAC GTACTGGGATGCCTCTCATC AAATATTACC TTGATGGCGA GAATACCCCT 540 CAGCTGTACA ATAATCACTTCTTCGCCAAG GGCGTGTCGG ACCCCTATAT GGTGAGCGTG 600 GCCAATGGCG GCACCGTGCCAGATGATGTT GTCGATAGTG TTGGAGAGAA GTTACAACAG 660 GCTTTCGGTT ATTACAAAGAGAAGCTTGCT GAGGACTTCG ACAAAGGGTT CGATGAGCTC 720 ATGCTCGTCG ACGACATGACCACTCGAGAG TACTTGAAGC GAGGCGGGCC GAAGGGAGAG 780 GCGCCCAAGT ATGACTTTTTCGCCATCCAG TGGATGGAGA CACAAAACAC TGGGACAAAC 840 CTGTTTGATC AGGCCTTTTCTGAAAGCGTC ATCGACTCGT TTGACTTTGA CAACCCGACA 900 AAGCCCGAAT GGTACTGCATCGAGGGAGGA ACATCGCTTT TGGTGGACGC CATGAAAGAA 960 ACCCTTGTCC ACAAGGTACAGAACAACAAG AGAGTTGATG CCATTTCCAT TGACTTGGAC 1020 GCTCCGGATG ATGGGAACATGTCGGTCAGG ATAGGCGGAA AGGATCACTC CGGATATAGC 1080 ACCGTCTTCA ACACCACCGCTCTGGGCTGC CTTGACCGCA TGGATCTGCG TGGTCTCAAC 1140 TTGCACCCTA CTCAGGCAGATGCCATTCGA TGTTTGCACT ATGACAACTC GACCAAGGTG 1200 GCTCTCAAGT TTAGCTACCCGTGGTGGATC AAGGACTGTG GCATCACTTG CGGTGGCGCG 1260 GCCTCGACTG ATCTACCTCTACGAACTTGC GTTTACCCAT CATACAACTT GGACGATACT 1320 GGTGAGGCTG TTCTGCTTGCCTCATACACT TGGTCTCAAG ATGCAACTCG CATTGGATCG 1380 TTGGTGAAGG ACGCTCCACCACAGCCGCCC AAGGAGGATG AGCTTGTCGA GCTGATCCTG 1440 CAGAACCTAG CCCGCCTGCACGCTGAGCAT ATGACCTACG AGAAGATTAA GGAGGCTTAC 1500 ACGGGCGTAT ATCACGCCTATTGCTGGGCT AATGATCCCA ATGTCGGTGG TGCTTTCGCC 1560 CTCTTCGGTC CCGGCCAGTTCAGCAATCTG TATCCATACC TGATGCGGCC AGCGGCGGGC 1620 GGCAAGTTCC ATATCGTCGGAGAGGCATCT AGTGTGCATC ACGCCTGGAT CATAGGGTCT 1680 TTGGAGAGCG CTTACACCGCTGTGTACCAG TTCTTGTACA AGTACAAGAT GTGGGATTAC 1740 TTGAGGTTGT TGTTGGAGCGCTGGCAGTAT GGTCTCCAGG AGTTAGAGAC GGGGAAGCAC 1800 GGTACGGCTC ATTTGCAGTTTATTCTAGGT TCACTTCCCA AGGAGTACCA GGTGAAGATT 1860 TAATAAGAAT TCGGTACTACGGC 1883 617 amino acids amino acid Not Relevant linear 46 Met Asp AsnVal Asp Phe Ala Glu Ser Val Arg Thr Arg Trp Ala Arg 1 5 10 15 Arg LeuIle Arg Glu Lys Val Ala Lys Glu Leu Asn Ile Leu Thr Glu 20 25 30 Arg LeuGly Glu Val Pro Gly Ile Pro Pro Pro Asn Glu Gly Arg Phe 35 40 45 Leu GlyGly Gly Tyr Ser His Asp Asn Leu Pro Ser Asp Pro Leu Tyr 50 55 60 Ser SerIle Lys Pro Ala Leu Leu Lys Glu Ala Pro Arg Ala Glu Glu 65 70 75 80 GluLeu Pro Pro Arg Lys Val Cys Ile Val Gly Ala Gly Val Ser Gly 85 90 95 LeuTyr Ile Ala Met Ile Leu Asp Asp Leu Lys Ile Pro Asn Leu Thr 100 105 110Tyr Asp Ile Phe Glu Ser Ser Ser Arg Thr Gly Gly Arg Leu Tyr Thr 115 120125 His His Phe Thr Asp Ala Lys His Asp Tyr Tyr Asp Ile Gly Ala Met 130135 140 Arg Tyr Pro Asp Ile Pro Ser Met Lys Arg Thr Phe Asn Leu Phe Lys145 150 155 160 Arg Thr Gly Met Pro Leu Ile Lys Tyr Tyr Leu Asp Gly GluAsn Thr 165 170 175 Pro Gln Leu Tyr Asn Asn His Phe Phe Ala Lys Gly ValSer Asp Pro 180 185 190 Tyr Met Val Ser Val Ala Asn Gly Gly Thr Val ProAsp Asp Val Val 195 200 205 Asp Ser Val Gly Glu Lys Leu Gln Gln Ala PheGly Tyr Tyr Lys Glu 210 215 220 Lys Leu Ala Glu Asp Phe Asp Lys Gly PheAsp Glu Leu Met Leu Val 225 230 235 240 Asp Asp Met Thr Thr Arg Glu TyrLeu Lys Arg Gly Gly Pro Lys Gly 245 250 255 Glu Ala Pro Lys Tyr Asp PhePhe Ala Ile Gln Trp Met Glu Thr Gln 260 265 270 Asn Thr Gly Thr Asn LeuPhe Asp Gln Ala Phe Ser Glu Ser Val Ile 275 280 285 Asp Ser Phe Asp PheAsp Asn Pro Thr Lys Pro Glu Trp Tyr Cys Ile 290 295 300 Glu Gly Gly ThrSer Leu Leu Val Asp Ala Met Lys Glu Thr Leu Val 305 310 315 320 His LysVal Gln Asn Asn Lys Arg Val Asp Ala Ile Ser Ile Asp Leu 325 330 335 AspAla Pro Asp Asp Gly Asn Met Ser Val Arg Ile Gly Gly Lys Asp 340 345 350His Ser Gly Tyr Ser Thr Val Phe Asn Thr Thr Ala Leu Gly Cys Leu 355 360365 Asp Arg Met Asp Leu Arg Gly Leu Asn Leu His Pro Thr Gln Ala Asp 370375 380 Ala Ile Arg Cys Leu His Tyr Asp Asn Ser Thr Lys Val Ala Leu Lys385 390 395 400 Phe Ser Tyr Pro Trp Trp Ile Lys Asp Cys Gly Ile Thr CysGly Gly 405 410 415 Ala Ala Ser Thr Asp Leu Pro Leu Arg Thr Cys Val TyrPro Ser Tyr 420 425 430 Asn Leu Asp Asp Thr Gly Glu Ala Val Leu Leu AlaSer Tyr Thr Trp 435 440 445 Ser Gln Asp Ala Thr Arg Ile Gly Ser Leu ValLys Asp Ala Pro Pro 450 455 460 Gln Pro Pro Lys Glu Asp Glu Leu Val GluLeu Ile Leu Gln Asn Leu 465 470 475 480 Ala Arg Leu His Ala Glu His MetThr Tyr Glu Lys Ile Lys Glu Ala 485 490 495 Tyr Thr Gly Val Tyr His AlaTyr Cys Trp Ala Asn Asp Pro Asn Val 500 505 510 Gly Gly Ala Phe Ala LeuPhe Gly Pro Gly Gln Phe Ser Asn Leu Tyr 515 520 525 Pro Tyr Leu Met ArgPro Ala Ala Gly Gly Lys Phe His Ile Val Gly 530 535 540 Glu Ala Ser SerVal His His Ala Trp Ile Ile Gly Ser Leu Glu Ser 545 550 555 560 Ala TyrThr Ala Val Tyr Gln Phe Leu Tyr Lys Tyr Lys Met Trp Asp 565 570 575 TyrLeu Arg Leu Leu Leu Glu Arg Trp Gln Tyr Gly Leu Gln Glu Leu 580 585 590Glu Thr Gly Lys His Gly Thr Ala His Leu Gln Phe Ile Leu Gly Ser 595 600605 Leu Pro Lys Glu Tyr Gln Val Lys Ile 610 615 31 base pairs nucleicacid single linear 47 CCCAGATCTA TATTTGCAAA CATGGACAAT G 31 33 basepairs nucleic acid single linear 48 GGGTCTAGAC TAACAAACAT CACACTTTCT ATG33 415 base pairs nucleic acid single linear 49 TCTAGAGGAT CAGCATGGCGCCCACCGTGA TGATGGCCTC GTCGGCCACC GCCGTCGCTC 60 CGTTCCTGGG GCTCAAGTCCACCGCCAGCC TCCCCGTCGC CCGCCGCTCC TCCAGAAGCC 120 TCGGCAACGT CAGCAACGGCGGAAGGATCC GGTGCATGCA GGTAACAAAT GCATCCTAGC 180 TAGTAGTTCT TTGCATTGCAGCAGCTGCAG CTAGCGAGTT AGTAATAGGA AGGGAACTGA 240 TGATCCATGC ATGGACTGATGTGTGTTGCC CATCCCATCC CATCCCATTT CCCAAACGAA 300 CCGAAAACAC CGTACTACGTGCAGGTGTGG CCCTACGGCA ACAAGAAGTT CGAGACGCTG 360 TCGTACCTGC CGCCGCTGTCGACCGGCGGG CGCATCCGCT GCATGCAGGC CATGG 415 1950 base pairs nucleic acidsingle linear 50 CCCGTTACTA CCATGGCTGC TATGGACAAT GTTGACTTTG CTGAATCTGTCCGAACCCGC 60 TGGGCGAGGC GACTCATTCG TGAGAAGGTC GCCAAGGAAC TCAACATTCTAACCGAAAGA 120 CTTGGTGAGG TGCCCGGCAT CCCTCCTCCA AATGAAGGCA GGTTCCTGGGCGGCGGCTAC 180 TCTCACGACA ATCTACCGTC TGATCCCCTC TATTCCAGCA TTAAGCCGGCTCTTCTAAAG 240 GAGGCTCCTC GAGCAGAAGA GGAACTGCCG CCTCGAAAGG TGTGCATCGTAGGCGCTGGT 300 GTTTCCGGCC TCTACATAGC CATGATTTTG GATGATTTGA AAATCCCAAATCTCACTTAC 360 GACATCTTCG AATCCAGTTC CAGAACTGGT GGCCGTTTGT ATACGCACCATTTCACCGAC 420 GCCAAGCATG ACTATTACGA CATTGGTGCT ATGCGATATC CTGACATCCCCAGCATGAAA 480 CGTACCTTTA ACCTGTTTAA ACGTACTGGG ATGCCTCTCA TCAAATATTACCTTGATGGC 540 GAGAATACCC CTCAGCTGTA CAATAATCAC TTCTTCGCCA AGGGCGTGTCGGACCCCTAT 600 ATGGTGAGCG TGGCCAATGG CGGCACCGTG CCAGATGATG TTGTCGATAGTGTTGGAGAG 660 AAGTTACAAC AGGCTTTCGG TTATTACAAA GAGAAGCTTG CTGAGGACTTCGACAAAGGG 720 TTCGATGAGC TCATGCTCGT CGACGACATG ACCACTCGAG AGTACTTGAAGCGAGGCGGG 780 CCGAAGGGAG AGGCGCCCAA GTATGACTTT TTCGCCATCC AGTGGATGGAGACACAAAAC 840 ACTGGGACAA ACCTGTTTGA TCAGGCCTTT TCTGAAAGCG TCATCGACTCGTTTGACTTT 900 GACAACCCGA CAAAGCCCGA ATGGTACTGC ATCGAGGGAG GAACATCGCTTTTGGTGGAC 960 GCCATGAAAG AAACCCTTGT CCACAAGGTA CAGAACAACA AGAGAGTTGATGCCATTTCC 1020 ATTGACTTGG ACGCTCCGGA TGATGGGAAC ATGTCGGTCA GGATAGGCGGAAAGGATCAC 1080 TCCGGATATA GCACCGTCTT CAACACCACC GCTCTGGGCT GCCTTGACCGCATGGATCTG 1140 CGTGGTCTCA ACTTGCACCC TACTCAGGCA GATGCCATTC GATGTTTGCACTATGACAAC 1200 TCGACCAAGG TGGCTCTCAA GTTTAGCTAC CCGTGGTGGA TCAAGGACTGTGGCATCACT 1260 TGCGGTGGCG CGGCCTCGAC TGATCTACCT CTACGAACTT GCGTTTACCCATCATACAAC 1320 TTGGACGATA CTGGTGAGGC TGTTCTGCTT GCCTCATACA CTTGGTCTCAAGATGCAACT 1380 CGCATTGGAT CGTTGGTGAA GGACGCTCCA CCACAGCCGC CCAAGGAGGATGAGCTTGTC 1440 GAGCTGATCC TGCAGAACCT AGCCCGCCTG CACGCTGAGC ATATGACCTACGAGAAGATT 1500 AAGGAGGCTT ACACGGGCGT ATATCACGCC TATTGCTGGG CTAATGATCCCAATGTCGGT 1560 GGTGCTTTCG CCCTCTTCGG TCCCGGCCAG TTCAGCAATC TGTATCCATACCTGATGCGG 1620 CCAGCGGCGG GCGGCAAGTT CCATATCGTC GGAGAGGCAT CTAGTGTGCATCACGCCTGG 1680 ATCATAGGGT CTTTGGAGAG CGCTTACACC GCTGTGTACC AGTTCTTGTACAAGTACAAG 1740 ATGTGGGATT ACTTGAGGTT GTTGTTGGAG CGCTGGCAGT ATGGTCTCCAGGAGTTAGAG 1800 ACGGGGAAGC ACGGTACGGC TCATTTGCAG TTTATTCTAG GTTCACTTCCCAAGGAGTAC 1860 CAGGTGAAGA TTTAAAGCGA AAGAGGTACT ACGGCATGGA GACAATTTTGGGTAGAGATT 1920 CTAGTATTCC AGCAGTTTCA TAATAATAAG 1950 34 base pairsnucleic acid single linear 51 CCCGTTACTA CCATGGCTGC TATGGACAAT GTTG 3434 base pairs nucleic acid single linear 52 CTAACAAACA GAATTCTTATTATTATGAAA CTGC 34 193 base pairs nucleic acid single linear 53TCTAGACCGG GCATGGCGTC CCGCCGGGTC TTCTCGTCCA TCCTTCGCTC TGCCTCTCGC 60ATTCGCTCCG CCTCACCGTC CCCATGCCCG CGTGCGCCGC TCCACCACCG CCCGTCCCCC 120GCGGGCTTCA TACTCAACCG TGTAGCCGCC TACGCCTCCT CCGCCACGGC CCAGGCGGCA 180CCTGCCATGG CGC 193 34 amino acids amino acid Not Relevant linear 54 MetGlu Asp Ala Ala Ala Arg Arg Met Arg Leu Ala Ser His Leu Arg 1 5 10 15Pro Pro Ala Ser Gln Met Glu Glu Ser Pro Leu Leu Arg Gly Ser Asn 20 25 30Cys Arg 107 base pairs nucleic acid single linear 55 CCATGGAGGACGCAGCAGCA AGGCGGATGG AGAGGCTCGC CTCCCACCTC CGCCCGCCCG 60 CTTCTCAGATGGAGGAATCA CCCCTCCTGA GGGGCTCCAA TTGCCGG 107 617 amino acids amino acidNot Relevant linear 56 Met Asp Asn Val Asp Phe Ala Glu Ser Val Arg ThrArg Trp Ala Arg 1 5 10 15 Arg Leu Ile Arg Glu Lys Val Ala Lys Glu LeuAsn Ile Leu Thr Glu 20 25 30 Arg Leu Gly Glu Val Pro Gly Ile Pro Pro ProAsn Glu Gly Arg Phe 35 40 45 Leu Gly Gly Gly Tyr Ser His Asp Asn Leu ProSer Asp Pro Leu Tyr 50 55 60 Ser Ser Ile Lys Pro Ala Leu Leu Gly Gly GlyGly Xaa Xaa Xaa Xaa 65 70 75 80 Xaa Xaa Gly Gly Gly Gly Val Cys Ile ValGly Ala Gly Val Ser Gly 85 90 95 Leu Tyr Ile Ala Met Ile Leu Asp Asp LeuLys Ile Pro Asn Leu Thr 100 105 110 Tyr Asp Ile Phe Glu Ser Ser Ser ArgThr Gly Gly Arg Leu Tyr Thr 115 120 125 His His Phe Thr Asp Ala Lys HisAsp Tyr Tyr Asp Ile Gly Ala Met 130 135 140 Arg Tyr Pro Asp Ile Pro SerMet Lys Arg Thr Phe Asn Leu Phe Lys 145 150 155 160 Arg Thr Gly Met ProLeu Ile Lys Tyr Tyr Leu Asp Gly Glu Asn Thr 165 170 175 Pro Gln Leu TyrAsn Asn His Phe Phe Ala Lys Gly Val Ser Asp Pro 180 185 190 Tyr Met ValSer Val Ala Asn Gly Gly Thr Val Pro Asp Asp Val Val 195 200 205 Asp SerVal Gly Glu Lys Leu Gln Gln Ala Phe Gly Tyr Tyr Lys Glu 210 215 220 LysLeu Ala Glu Asp Phe Asp Lys Gly Phe Asp Glu Leu Met Leu Val 225 230 235240 Asp Asp Met Thr Thr Arg Glu Tyr Leu Lys Arg Gly Gly Pro Lys Gly 245250 255 Glu Ala Pro Lys Tyr Asp Phe Phe Ala Ile Gln Trp Met Glu Thr Gln260 265 270 Asn Thr Gly Thr Asn Leu Phe Asp Gln Ala Phe Ser Glu Ser ValIle 275 280 285 Asp Ser Phe Asp Phe Asp Asn Pro Thr Lys Pro Glu Trp TyrCys Ile 290 295 300 Glu Gly Gly Thr Ser Leu Leu Val Asp Ala Met Lys GluThr Leu Val 305 310 315 320 His Lys Val Gln Asn Asn Lys Arg Val Asp AlaIle Ser Ile Asp Leu 325 330 335 Asp Ala Pro Asp Asp Gly Asn Met Ser ValArg Ile Gly Gly Lys Asp 340 345 350 His Ser Gly Tyr Ser Thr Val Phe AsnThr Thr Ala Leu Gly Cys Leu 355 360 365 Asp Arg Met Asp Leu Arg Gly LeuAsn Leu His Pro Thr Gln Ala Asp 370 375 380 Ala Ile Arg Cys Leu His TyrAsp Asn Ser Thr Lys Val Ala Leu Lys 385 390 395 400 Phe Ser Tyr Pro TrpTrp Ile Lys Asp Cys Gly Ile Thr Cys Gly Gly 405 410 415 Ala Ala Ser ThrAsp Leu Pro Leu Arg Thr Cys Val Tyr Pro Ser Tyr 420 425 430 Asn Leu AspAsp Thr Gly Glu Ala Val Leu Leu Ala Ser Tyr Thr Trp 435 440 445 Ser GlnAsp Ala Thr Arg Ile Gly Ser Leu Val Lys Asp Ala Pro Pro 450 455 460 GlnPro Pro Lys Glu Asp Glu Leu Val Glu Leu Ile Leu Gln Asn Leu 465 470 475480 Ala Arg Leu His Ala Glu His Met Thr Tyr Glu Lys Ile Lys Glu Ala 485490 495 Tyr Thr Gly Val Tyr His Ala Tyr Cys Trp Ala Asn Asp Pro Asn Val500 505 510 Gly Gly Ala Phe Ala Leu Phe Gly Pro Gly Gln Phe Ser Asn LeuTyr 515 520 525 Pro Tyr Leu Met Arg Pro Ala Ala Gly Gly Lys Phe His IleVal Gly 530 535 540 Glu Ala Ser Ser Val His His Ala Trp Ile Ile Gly SerLeu Glu Ser 545 550 555 560 Ala Tyr Thr Ala Val Tyr Gln Phe Leu Tyr LysTyr Lys Met Trp Asp 565 570 575 Tyr Leu Arg Leu Leu Leu Glu Arg Trp GlnTyr Gly Leu Gln Glu Leu 580 585 590 Glu Thr Gly Lys His Gly Thr Ala HisLeu Gln Phe Ile Leu Gly Ser 595 600 605 Leu Pro Lys Glu Tyr Gln Val LysIle 610 615 617 amino acids amino acid Not Relevant linear 57 Met AspAsn Val Asp Phe Ala Glu Ser Val Arg Thr Arg Trp Ala Arg 1 5 10 15 ArgLeu Ile Arg Glu Lys Val Ala Lys Glu Leu Asn Ile Leu Thr Glu 20 25 30 ArgLeu Gly Glu Val Pro Gly Ile Pro Pro Pro Asn Glu Gly Arg Phe 35 40 45 LeuGly Gly Gly Tyr Ser His Asp Asn Leu Pro Ser Asp Pro Leu Tyr 50 55 60 SerSer Ile Gly Gly Gly Ser Gly Gly Xaa Xaa Xaa Xaa Xaa Xaa Gly 65 70 75 80Gly Gly Pro Pro Arg Lys Val Cys Ile Val Gly Ala Gly Val Ser Gly 85 90 95Leu Tyr Ile Ala Met Ile Leu Asp Asp Leu Lys Ile Pro Asn Leu Thr 100 105110 Tyr Asp Ile Phe Glu Ser Ser Ser Arg Thr Gly Gly Arg Leu Tyr Thr 115120 125 His His Phe Thr Asp Ala Lys His Asp Tyr Tyr Asp Ile Gly Ala Met130 135 140 Arg Tyr Pro Asp Ile Pro Ser Met Lys Arg Thr Phe Asn Leu PheLys 145 150 155 160 Arg Thr Gly Met Pro Leu Ile Lys Tyr Tyr Leu Asp GlyGlu Asn Thr 165 170 175 Pro Gln Leu Tyr Asn Asn His Phe Phe Ala Lys GlyVal Ser Asp Pro 180 185 190 Tyr Met Val Ser Val Ala Asn Gly Gly Thr ValPro Asp Asp Val Val 195 200 205 Asp Ser Val Gly Glu Lys Leu Gln Gln AlaPhe Gly Tyr Tyr Lys Glu 210 215 220 Lys Leu Ala Glu Asp Phe Asp Lys GlyPhe Asp Glu Leu Met Leu Val 225 230 235 240 Asp Asp Met Thr Thr Arg GluTyr Leu Lys Arg Gly Gly Pro Lys Gly 245 250 255 Glu Ala Pro Lys Tyr AspPhe Phe Ala Ile Gln Trp Met Glu Thr Gln 260 265 270 Asn Thr Gly Thr AsnLeu Phe Asp Gln Ala Phe Ser Glu Ser Val Ile 275 280 285 Asp Ser Phe AspPhe Asp Asn Pro Thr Lys Pro Glu Trp Tyr Cys Ile 290 295 300 Glu Gly GlyThr Ser Leu Leu Val Asp Ala Met Lys Glu Thr Leu Val 305 310 315 320 HisLys Val Gln Asn Asn Lys Arg Val Asp Ala Ile Ser Ile Asp Leu 325 330 335Asp Ala Pro Asp Asp Gly Asn Met Ser Val Arg Ile Gly Gly Lys Asp 340 345350 His Ser Gly Tyr Ser Thr Val Phe Asn Thr Thr Ala Leu Gly Cys Leu 355360 365 Asp Arg Met Asp Leu Arg Gly Leu Asn Leu His Pro Thr Gln Ala Asp370 375 380 Ala Ile Arg Cys Leu His Tyr Asp Asn Ser Thr Lys Val Ala LeuLys 385 390 395 400 Phe Ser Tyr Pro Trp Trp Ile Lys Asp Cys Gly Ile ThrCys Gly Gly 405 410 415 Ala Ala Ser Thr Asp Leu Pro Leu Arg Thr Cys ValTyr Pro Ser Tyr 420 425 430 Asn Leu Asp Asp Thr Gly Glu Ala Val Leu LeuAla Ser Tyr Thr Trp 435 440 445 Ser Gln Asp Ala Thr Arg Ile Gly Ser LeuVal Lys Asp Ala Pro Pro 450 455 460 Gln Pro Pro Lys Glu Asp Glu Leu ValGlu Leu Ile Leu Gln Asn Leu 465 470 475 480 Ala Arg Leu His Ala Glu HisMet Thr Tyr Glu Lys Ile Lys Glu Ala 485 490 495 Tyr Thr Gly Val Tyr HisAla Tyr Cys Trp Ala Asn Asp Pro Asn Val 500 505 510 Gly Gly Ala Phe AlaLeu Phe Gly Pro Gly Gln Phe Ser Asn Leu Tyr 515 520 525 Pro Tyr Leu MetArg Pro Ala Ala Gly Gly Lys Phe His Ile Val Gly 530 535 540 Glu Ala SerSer Val His His Ala Trp Ile Ile Gly Ser Leu Glu Ser 545 550 555 560 AlaTyr Thr Ala Val Tyr Gln Phe Leu Tyr Lys Tyr Lys Met Trp Asp 565 570 575Tyr Leu Arg Leu Leu Leu Glu Arg Trp Gln Tyr Gly Leu Gln Glu Leu 580 585590 Glu Thr Gly Lys His Gly Thr Ala His Leu Gln Phe Ile Leu Gly Ser 595600 605 Leu Pro Lys Glu Tyr Gln Val Lys Ile 610 615 617 amino acidsamino acid Not Relevant linear 58 Met Asp Asn Val Asp Phe Ala Glu SerVal Arg Thr Arg Trp Ala Arg 1 5 10 15 Arg Leu Ile Arg Glu Lys Val AlaLys Glu Leu Asn Ile Leu Thr Glu 20 25 30 Arg Leu Gly Glu Val Pro Gly IlePro Pro Pro Asn Glu Gly Arg Phe 35 40 45 Leu Gly Gly Gly Tyr Ser His AspAsn Leu Pro Ser Asp Pro Leu Tyr 50 55 60 Ser Ser Ile Lys Pro Gly Gly GlyGly Xaa Xaa Xaa Xaa Xaa Xaa Gly 65 70 75 80 Gly Gly Pro Pro Arg Lys ValCys Ile Val Gly Ala Gly Val Ser Gly 85 90 95 Leu Tyr Ile Ala Met Ile LeuAsp Asp Leu Lys Ile Pro Asn Leu Thr 100 105 110 Tyr Asp Ile Phe Glu SerSer Ser Arg Thr Gly Gly Arg Leu Tyr Thr 115 120 125 His His Phe Thr AspAla Lys His Asp Tyr Tyr Asp Ile Gly Ala Met 130 135 140 Arg Tyr Pro AspIle Pro Ser Met Lys Arg Thr Phe Asn Leu Phe Lys 145 150 155 160 Arg ThrGly Met Pro Leu Ile Lys Tyr Tyr Leu Asp Gly Glu Asn Thr 165 170 175 ProGln Leu Tyr Asn Asn His Phe Phe Ala Lys Gly Val Ser Asp Pro 180 185 190Tyr Met Val Ser Val Ala Asn Gly Gly Thr Val Pro Asp Asp Val Val 195 200205 Asp Ser Val Gly Glu Lys Leu Gln Gln Ala Phe Gly Tyr Tyr Lys Glu 210215 220 Lys Leu Ala Glu Asp Phe Asp Lys Gly Phe Asp Glu Leu Met Leu Val225 230 235 240 Asp Asp Met Thr Thr Arg Glu Tyr Leu Lys Arg Gly Gly ProLys Gly 245 250 255 Glu Ala Pro Lys Tyr Asp Phe Phe Ala Ile Gln Trp MetGlu Thr Gln 260 265 270 Asn Thr Gly Thr Asn Leu Phe Asp Gln Ala Phe SerGlu Ser Val Ile 275 280 285 Asp Ser Phe Asp Phe Asp Asn Pro Thr Lys ProGlu Trp Tyr Cys Ile 290 295 300 Glu Gly Gly Thr Ser Leu Leu Val Asp AlaMet Lys Glu Thr Leu Val 305 310 315 320 His Lys Val Gln Asn Asn Lys ArgVal Asp Ala Ile Ser Ile Asp Leu 325 330 335 Asp Ala Pro Asp Asp Gly AsnMet Ser Val Arg Ile Gly Gly Lys Asp 340 345 350 His Ser Gly Tyr Ser ThrVal Phe Asn Thr Thr Ala Leu Gly Cys Leu 355 360 365 Asp Arg Met Asp LeuArg Gly Leu Asn Leu His Pro Thr Gln Ala Asp 370 375 380 Ala Ile Arg CysLeu His Tyr Asp Asn Ser Thr Lys Val Ala Leu Lys 385 390 395 400 Phe SerTyr Pro Trp Trp Ile Lys Asp Cys Gly Ile Thr Cys Gly Gly 405 410 415 AlaAla Ser Thr Asp Leu Pro Leu Arg Thr Cys Val Tyr Pro Ser Tyr 420 425 430Asn Leu Asp Asp Thr Gly Glu Ala Val Leu Leu Ala Ser Tyr Thr Trp 435 440445 Ser Gln Asp Ala Thr Arg Ile Gly Ser Leu Val Lys Asp Ala Pro Pro 450455 460 Gln Pro Pro Lys Glu Asp Glu Leu Val Glu Leu Ile Leu Gln Asn Leu465 470 475 480 Ala Arg Leu His Ala Glu His Met Thr Tyr Glu Lys Ile LysGlu Ala 485 490 495 Tyr Thr Gly Val Tyr His Ala Tyr Cys Trp Ala Asn AspPro Asn Val 500 505 510 Gly Gly Ala Phe Ala Leu Phe Gly Pro Gly Gln PheSer Asn Leu Tyr 515 520 525 Pro Tyr Leu Met Arg Pro Ala Ala Gly Gly LysPhe His Ile Val Gly 530 535 540 Glu Ala Ser Ser Val His His Ala Trp IleIle Gly Ser Leu Glu Ser 545 550 555 560 Ala Tyr Thr Ala Val Tyr Gln PheLeu Tyr Lys Tyr Lys Met Trp Asp 565 570 575 Tyr Leu Arg Leu Leu Leu GluArg Trp Gln Tyr Gly Leu Gln Glu Leu 580 585 590 Glu Thr Gly Lys His GlyThr Ala His Leu Gln Phe Ile Leu Gly Ser 595 600 605 Leu Pro Lys Glu TyrGln Val Lys Ile 610 615

What is claimed is:
 1. A composition for controlling insect infestationof plants, said composition comprising: a lysine oxidase enzyme; and aprotein comprising an amino acid sequence as set forth in SEQ ID NO:41wherein said composition is ingested by an insect.
 2. The composition ofclaim 1, wherein said composition exhibits coleopteran insecticidalactivity.
 3. The composition of claim 2, wherein said coleopteraninsecticidal activity is effective in controlling coleopteran speciesselected from the group consisting of Diabrotica, Melanotus,Leptinotarsa, and Anthonomus.
 4. The composition of claim 3, whereinsaid coleopteran insecticidal activity is effective in controllinginsects selected from the group consisting of boll weevil (BWV), cornrootworm (CRW), wireworm (WW), and Colorado potato beetle (CPB).
 5. Thecomposition of claim 1, wherein said lysine oxidase enzyme and saidprotein are present respectively in a molar ratio of about 100:1 toabout 1:1.
 6. The composition of claim 1, wherein said lysine oxidaseenzyme and said protein are present respectively in a molar ratio ofabout 10:1 to about 1:1.
 7. The composition of claim 1, wherein saidprotein and said lysine oxidase enzyme are present respectively in amolar ratio of about 100:1 to about 1:1.
 8. The composition of claim 1,wherein said protein and said lysine oxidase enzyme are presentrespectively in a molar ratio of about 10:1 to about 1:1.
 9. A method ofcontrolling insect infestation of plants comprising providing acomposition comprising: a lysine oxidase enzyme; and a proteincomprising an amino acid sequence as set forth in SEQ ID NO:41; whereinsaid composition is ingested by an insect.
 10. The method of claim 9,wherein said composition exhibits coleopteran insecticidal activity. 11.The method of claim 10, wherein said coleopteran insecticidal activityis effective in controlling coleopteran species selected from the groupconsisting of Diabrotica, Melanotus, Leptinotarsa, and Anthonomus. 12.The method of claim 11, wherein said coleopteran insecticidal activityis effective in controlling insects selected from the group consistingof boll weevil (BWV), corn rootworm (CRW), wireworm (WW), and Coloradopotato beetle (CPB).
 13. The method of claim 9, wherein said lysineoxidase enzyme and said protein are present respectively in said mixturein a molar ratio of about 100:1 to about 1:1.
 14. The method of claim 9,wherein said lysine oxidase enzyme and said protein are presentrespectively in said composition in a molar ratio of about 10:1 to about1:1.
 15. The method of claim 9, wherein said protein and said lysineoxidase enzyme are present respectively in said composition in a molarratio of about 100:1 to about 1:1.
 16. The method of claim 9, whereinsaid protein and said lysine oxidase enzyme are present respectively insaid composition in a molar ratio of about 10:1 to about 1:1.